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 ABC Transporters
A Goffeau, Université Catholique de Louvain, Louvain-la-Neuve, Belgium
B De Hertogh, Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium
ã 2013 Elsevier Inc. All rights reserved.

This article is a revision of the previous edition article by André Goffeau, Benoı̂t De Hertogh, and Philippe V. Baret, volume 1, pp. 1–5, ã 2004, Elsevier Inc.

Glossary and which is often activated during cancer

ABC1 Specific small amino acid sequence which is a chemotherapy.
specific signature of ABC proteins. Transmembrane span (TMS) and transmembrane
C-, D-, H-, P-, and Q-loop Loops are located at the junction domain (TMD) The existence of the transmembrane
of a-helices and are represented by conserved amino acid span is predicted by frequency of hydrophobic residues in a
residue in one letter code: C is cysteine, D is aspartate, reading window of about 17 amino acids. In ABC
H is histidine, P is proline, and Q is asparagine. transporters, the transmembrane domains usually
NBD Nucleotide-binding domain of about 100 amino comprise six contigous transmembrane spans.
acids which is characterized by a Walker A and Walker Transporter classification (TC) Classification of
B motif. over 1000 transporter families, developed by Milton
Pdr5p The major yeast ABC transporter involved in Saier based on a combination of mechanistic and
pleiotropic drug resistance including the azole-antifungals. phylogenetic criteria.
P-gp The major ABC transporter (glycoprotein) Walker A and B Small consensus of amino acid sequences
involved in mammalian multidrug resistance involved in ATP binding.

The ABCs constitute the largest family of proteins. They are often associate with two TMDs either as four single subunits
present in all living species from Archaea to Homo sapiens. encoded within the same operon or in various combinations of
They make up to 4% of the full genome complement of bacteria fused subunits (Figure 2). Association of other proteins may
such as Escherichia coli or Bacillus subtilis. Each eukaryote occur. The most prominent associated bacterial protein is the
genome contains several dozens of ABC proteins. They are periplasmic solute-binding receptor, which in Gram-negative
recognized by a consensus nucleotide-binding domain (NBD) bacteria is found in the periplasm, and in Gram-positive bacte-
of approximately 100 amino acids which comprises a series ria is often a lipoprotein bound to the external membrane
of structural and sequence motifs illustrated in Figure 1: the surface via electrostatic interactions. The three domains of the
Walker A motif, the ABC1 signature, the Walker B motif, bacterial ABC uptake transporters, namely the periplasmic-
the Helical domain, and the P, Q-, C-, D-, and H-loops which binding receptor, the cytoplasmic NBD, and the membrane
are structural junctions between a-helices marked by a con- TMD, are believed to have arisen from a common ancestral
served proline, asparagine, cysteine, aspartate, or histidine resi- ABC transporter in which these three proteins were already
due. The ABC proteins catalyze a wide variety of physiological present. However, during evolution the sequence of the peri-
functions, many but not all of which are related to transport. plasmic solute-binding receptors diverged more rapidly than
We wish to describe the major physiological and biochemical that of the TMDs while that of NBDs remained the least diver-
functions as well as the structural properties of some of the best- gent. Thus, all NBDs are homologous, but this is not true for
known ABC transporters using as examples the multidrug the TMDs or the receptors.
exporters Sav1866 from Staphylococcus aureus, Pdr5p from In eukaryotes, two TMDs and two NBDs are often, but
Saccharomyces cerevisiae, and P-gp from H. sapiens. not always, associated in one single molecule called ‘full-
sized ABC transporter’. The topological relation between
NBDs and TMDs is variable (Figure 3); additional TM spans
Topology
occur in some systems as well as extracytoplasmic domains
of presumed regulatory function.
Each ABC transporter molecule contains, or is associated to,
one or two cytoplasmic adenosine triphosphate (ATP)-binding
domains named NBDs and one or two transmembrane Phylogeny
domains (TMDs). Each TMD comprises usually six transmem-
brane spans (TMSs) separated by the three extracytoplasmic The different families of ABC proteins transport a wide variety
loops ECL1, ECL2, and ECL3 and the two intracytoplasmic of substrates against their concentration gradient using the
loops ICL2 and ICL3. Association of one TMD to one NBD energy of ATP hydrolysis carried out by NBD. In bacteria, the
results in a half-size ABC transporter, which functions as transported substrates are either imported in or exported out
homo- or heterodimers so that the minimal functional organi- of the cell. In eukaryotes, only extracytoplasmic exporters
zation of an ABC transporter is considered to be TMD–NBD/ transporting substrates either out of the cell or into organelles
TMD–NBD or NBD–TMD/NBD–TMD. In bacteria, two NBDs are known. Within the ABC transporters, a total of 173

7
8 Bioenergetics | ABC Transporters

Out
NBD

In C N Stalk
N C
NBD1 NBD2 NBD1 NBD2

TMD

N NBD1 NBD2 Figure 3 Reconstructed structure of the purified yeast Pdr5p

C reconstituted in lipid-containing particles and observed by cryo electron
microscopy.

This family is not detected in the animal kingdom. Con-

C N versely, the large human and mouse ABCA family identified
N C in 1994 by Giovanna Chimini is not represented in fungal
NBD NBD
genomes.
Figure 1 Example of topological relations between nucleotide-binding
domains (NBDs) and transmembrane domains (TMDs) in full-sized
and half-sized ABC transporters. Function and Diseases

The ABC transporters from Archaea, Gram-negative, and

Gram-positive plasma membranes are widely different in
Out
their organization and composition. The variety of substrates
Periplasmic-binding transported: sugars, amino acids, lipids, ions, polysaccharides,
receptor peptides, proteins, toxins, drugs, antibiotics, xenobiotics, and
other metabolites is reflected by the divergence of the periplas-
mic sensor and that of the TMD which control the specificity
Substrate
and the direction (efflux or influx) of transport.
Even if all eukaryotic ABC transporters are effluxers that
TMD1 TMD2 comprise each a TMD fused to NBD, some of them are not
Transmembrane domain
directly involved in moving substrates. For instance, in the
cystic fibrosis transmembrane regulator (CFTR) and in the sul-
Cytoplasmic nucleotide- fonylurea receptor (SUR), the hydrolysis of ATP appears to be
In NBD1 NBD2 binding domain linked to the regulation of opening and closing of ion channels
carried by the ABC protein itself or other proteins .The presence
of NBD and TMD in all ABC transporters, however, suggests
Figure 2 Example of protein subunits constituting bacterial ABC
influx transporters.
that a basic coupling mechanism exists for efflux and influx
whatever the transported substrate. However, distantly related
proteins utilize an NBD to drive diverse nontransport processes
such as DNA repair or protein elongation or regulation of
phylogenetic families have been identified so far and classified RNAse activities.
within the transporter classification (TC) system developed by One strong impetus for the study of mammalian ABC trans-
Milton Saier in San Diego. These families generally correlate porters is their involvement in human diseases. Many mende-
with substrate specificity and comprise both efflux and influx lian diseases and complex genetic disorders are caused by
transporters. ABC transporters including cystic fibrosis, adrenoleukodystro-
Since 1995, the in silico analysis of hundreds of full genome phy, Stargardt disease, Tangier disease, immune deficiencies,
sequences has allowed the identification of thousands of new progressive familial intrahepatic cholestasis, Dublin–Johnson
ABC proteins. For instance, within the 49 ABC proteins identi- syndrome, Pseudoxanthoma elasticum, persistent hyperinsuli-
fied in the human genome, five major families of transporters nemic hypoglycemia of infancy due to focal adenomatous
have been identified and named ABCA (or ABC1), ABCB (or hyperplasia, X-linked sideroblastosis and anemia, age-related
MDR including the famous P-gp described below), ABCC macular degeneration, familial hypoapoproteinemia, fundus
(or MRP), ABCD (or ALD), and ABCG (or WHITE). This clas- flavimaculatis, retinitis pigmentosum, and cone rod dystrophy.
sification overlaps with the TC. Frequent cases of resistance to plant, fungal, or mammalian
S. cerevisiae genome contains 32 ABC proteins among pathogens and to chemotherapic treatments of cancers often
which 22 are associated to TMDs. The largest yeast ABC family result from induction or activation mutations of pleiotropic
is Pdrp identified in 1997 by Anabelle Decottignies and André drug resistance (Pdrp), multiple drug resistance (MDR), or
Goffeau and shown later to be present in all fungi and plants. multidrug resistance-related protein (MRP) transporters. Basic
 Bioenergetics | ABC Transporters 9

studies of human, plants, or pathogen ABC transporters would the transmembrane spans 6 and 7 in BtuCD, or through the ICL3
greatly benefit from heterologous expression of human ABC and ICL 6 in Sav1866.
transporter genes in yeast or other cells but this technology is A clear contribution of the X-ray structures is the observa-
not fully satisfactory yet. Meanwhile, knockout technology in tion that two molecules of ATP are bound at the interface of the
the mouse allows physiological analysis of the mammalian two NBDs. Each nucleotide-binding site comprises a Walker A
transporters. motif from one monomer and the ABC1 C motif from the
other monomer in a head-to-tail arrangement of the two inter-
acting NBD monomers.
Structure and Biochemical Mechanism As the folds of the various ABC transporters are highly
variable we focus on some basic structural properties of the
In 2010, over a dozen high-resolutive X-ray structures of func- bacterial Sav1866 and the mammalian P-gp multidrug drug
tional ABC transporters are available and have contributed to exporters which have been reported, respectively, by Kaspar
the emergence of a consensus view of the catalytic mechanism Locher in 2006 and Goffrey Chang in 2009.
of transport. Recent analyses at the electron microscopy level of The homodimeric Sav1866 transporter has two identical
purified bacterial (BmrA from B. subtilis) and a fungal (Pdr5p subunits, each comprising a NBD fused to a TMD made of six
from S. cerevisiae) drug efflux ABC transporter came to a transmembrane a-helices . It exports a wide series of unrelated
remarkably coherent set of conclusions. In both cases, the drugs such as the anticancer compounds doxorubicin and
basic structural unit seems to comprise four joining NBDs vinblastin. Two coupling a-helixes, located in ICL3 and ICL6,
that correspond to two full-size Pdr5p or four half-size BmrA. interact with grooves from the two NBDs. This structural motif
The NBD are related to the TMDs through four distinct stalks. is conserved in all ABC transporters but its amino acid
Each NBD is oriented at a fixed 90º angle relative to its neigh- sequence is not conserved. Individual TMS from the two
bor NBDs. This raises the possibility of concerted rotation TMDs embrace each other and form two symmetric membrane
movements of the NBDs implying a certain flexibility of the bundles each made of six mixed TMS comprising two mem-
stalks. No intramolecular or no intramembrane pores were brane a-helices from one TMD and four membrane a-helices
observed even though there is room (or chamber) between from the other TMD. A central cavity was identified between
the four stalks that join together at their NBD tips (Figure 4). the TMDs. As it faces outward, it was interpreted to represent
The nature of the stalks, TMD and NBD, was further identi- an open drug extrusion pocket.
fied by the analysis of the well-resolved structure of complete P-gp is a full size NBD–TMD–NBD–TMD transporter which
dimeric ABC bacterial and eukaryote transporters such as is responsible for multiple drug resistance of mouse and
those obtained for the phospholipid flippase MsbA from human cell and to which has been attributed failure of chemo-
E. coli and Staphylococcus typhimurum, the vitamin B12 importer treated cancer patients. The mouse P-gp was expressed in the
BtuCD from E. coli, the importer of metal-chelate HI1470/1 yeast Pichia pastoris, purified and crystallized. This 3.8 Å struc-
from Haemophilus influenzae, the drug exporter Sav1866 from ture is a nucleotide-free, inward conformation arranged in two
S. aureus, and the mammalian multidrug exporter P-gp. symmetric halves each comprising an NBD and a TM bundle.
The folds of the membrane and stalk domains were found Each TM bundle comprises six mixed TMs in which spans from
to be dissimilar in the different structures analyzed so far. the two TMDs embrace each other. A large drug-binding pocket
This may not be surprising taking into account the different of about 6000 cubic A located at the intersection of the two TM
species analyzed, the different cocrystallized ligands, the differ- bundles is open to both the inner membrane leaflet and the
ent numbers of TMS, and the different functions of the proteins cytoplasm. A total of 73 amino acids’ aromatic and hydropho-
analyzed. In particular, the communication between the NBD bic residues facing this cavity was accessible to solvent and
and the TMD was found to be variable and, for instance, is involved in the overlapping binding of three different ABC
carried out either through a long and complex intracytoplas- inhibitors. Only two amino acid residues are common to the
mic loop (ICL) in MsbA, through a short L-shaped ICL between three binding sites. Two portals are wide open to the inner

Consensus motifs in NBD

Helical
Walker A1 ABC 1 Walker B1
domain

P-loop Q-loop C-loop pro-loop D-loop H-loop

90–110 aa

Figure 4 The consensus adenosine triphosphate (ATP)-binding region of a typical ABC protein is made of approximately 100 amino acids (aa),
including the Walker A motif, the ABC1 signature, the Walker B motif, the helical domain, and the P-, Q-, C-, D-, and H-loops (see section Glossary).
10 Bioenergetics | ABC Transporters

phospholipid bilayer and allow entry of hydrophobic drugs MDR family of mammalian ABC exporters may have mechan-
which penetrate the membrane by passive diffusion. isms partially different from that of the Pdrp family of plant
and fungal multidrug transporters. Obviously, the resolution
of more complex mechanisms such as those involved in the
gated choride flux of CFTR, the regulation of the potassium
Transport Mechanism
channel of SUR1, or the loading of antigenic peptide by TAP1
will have to wait until more structural and biochemical infor-
Recent structural observations have led to the disposal of the
mation become available.
early hypothetic mechanism of alternating ATP-binding sites
during the drug transport cycle. Instead, a consensus emerges
favoring a rather simple alternating outward–inward access
mechanism by which binding and hydrolysis of ATP by the Substrate Specificity of the Multidrug Exporters and
cytoplasmic NBD controls the binding and translocation of the Reversal Agents
ligand through the TMD (Figure 5). Ligand binding to a high-
affinity pocket formed by the TMDs induces a conformational One of the most intriguing contemporary biochemical pro-
change in the NBDs resulting in a higher affinity for ATP. Two blems is the characterization of the interactions of the TMDs
molecules of ATP each binding to both NBDs induce a close with their transported substrate. The most extraordinary fea-
dimeric conformation which promotes the extrusion of the ture in this context is the apparent lack of specificity of the yeast
ligand. ATP hydrolysis triggers the opening of the NBD dimer. Pdr5p and human Pgp that transports hundreds of different
Finally, phosphate and ADP are released and the ligand-free chemicals, apparently contradicting the famous key/slot con-
NBD dimer conformation is restored. cept described in all textbooks. It is now recognized that at least
This two-state alternating access mechanism is shared not three partly overlapping drug-binding sites operate in MDR,
only by eukaryotic drug exporters but also by bacterial impor- MRP, and Pdrp multidrug transporters and that drug binding
ters. This is shown by analyses of the type 1 bacterial importers depends on the hydrogen-bound acceptor properties, presence
such as the maltose (MalFGK), histidine (HisPQM), and of aromatic rings, and hydrophobicity properties as well as on
methionine (MetNId/l) importers in which low-affinity sub- the surface volume of the substrates.
strate-binding proteins mediate the delivery of substrates to the Since 1980, tremendous efforts have been made to identify
ATP-bound form and also of the type 2 bacterial importers, inhibitors that can reverse MDR in cancer cells or Pdrp in fungi.
such as the vitamin B12 (BtuCD) and metal-chelate (Hi1470/1) Over 30 compounds with cabalistic names and structures have
importers which involve a binding protein of high affinity been shown to inhibit the cancer-related efflux pumps ABCB1
for the substrate even in the absence of ATP. The delivery (MDR), ABCC1 (MRP), or ABCG2 (BCRP), or the fungal Pdrp
of substrates via a binding protein introduces an additional transporters.
conformational switch that is of appreciable structural and Such inhibitors may act directly by acting as pseudosub-
mechanistic divergences between type 1 and type 2 importers. strate, as competitive inhibitor of ATP binding or as a noncom-
The multiple conformation changes occurring during the petitive inhibitor acting at remote sites. They may also act
ATP-driven substrate transport from prokaryotic and eukary- indirectly on aspects of metabolism that affect efflux. The
otic transporters are far from being unraveled; however, at variety of identified drug pump inhibitors indicates that the
present, it is best to regard the importers and the exporters as development of efficient and specific inhibition of the antican-
having partly distinct molecular mechanisms. Moreover, the cer and antifungal pumps should be possible. However, no

Drug

TMD TMD TMD TMD

NBD NBD NBD NBD

ATP
Figure 5 The flip-flop hypothesis for drug transport. Coupling between TMDs and NBDs is assured trough conserved – a-helices located in ICLs.
The inward conformation of the TM bundles binds drugs in its cavity which is open both to the cytoplasm and to the inner phospholipid leaflet of the
membrane. Upon binding of ATP, the NBDs dimerize and set the outward conformation in which the drug-binding site is exposed to the external
milieu. ATP hydrolysis releases drugs, disrupts NBD dimerization, and resets the inward conformation.
 Bioenergetics | ABC Transporters 11

ABC pump inhibitor is yet used clinically. Obviously, many of ABC transporters in bacteria, fungi, plants, and animals have
pump inhibitors, such as the antimalarial quinine derivatives to be sorted out. Specific inhibitors of ABC drug importers and
or the immunodepressor FK506 are known to act on diverse exporters have to be screened for. The physiological mechan-
physiological functions. Moreover, ABC drug pumps seem isms of ABC-linked diseases have to be further studied in
to be involved in important physiological functions not related mouse knockouts. Systems prone to specific inhibition of
to drug efflux such as the development, differentiation, and ABC transporters expression by interfering RNA have to be
maturation of immune cells. This may explain the multiple explored. Genetic therapy of the ABC diseases has to be
secondary effects of the inhibitors. One may expect that until developed.
the molecular structure and mechanisms of drug efflux are fully
understood, the development of anti-MDR or Pdrp compounds
will remain inefficient and merely based on brute screening. The See also: Lipids Carbohydrates Membranes and Membrane
ball is clearly in the hand of scientists studying basic mechan- Proteins: Multidrug Resistance Membrane Proteins.
isms. However, some peculiar clinical treatments have been
considered. For instance, low concentrations of isobuprofen,
which is a potent anti-inflammatory drug, inhibits azole efflux
Further Reading
from Candida albicans, and has been proposed for combination
treatment of fungal infections with fluconazole. Aller SG, Yu J, Ward A, et al. (2009) Structure of P-glycoprotein reveals a molecular
In addition, alternative strategies such as regulating expres- basis for poly-specific drug binding. Science 323: 1718–1722.
sion of drug transporters or uptake of anticancer drugs are also Cannon RD, Holmes AR, Mason AB, et al. (2009) Efflux-mediated antifungal drug
resistance. Clinical Microbiology Reviews 22: 291–321.
being considered. Dean M (2002) The Human ATP-Binding Cassette (ABC) Transporter Superfamily,
Monograph. Bethesda, MD: NCBI, National Library of Medicine (US).
Decottignies A and Goffeau A (1997) Complete inventory of the yeast ABC proteins.
Conclusion Nature Genetics 15: 137–145.
Gottesman MM and Ambudkar SV (2001) Overview: ABC transporters and human
disease. Journal of Bioenergetics and Biomembranes 33: 453–458.
The next frontiers in the study of ABC transporters are chal- Gutmann DA, Ward A, Urbatsch IL, Chang G, and van Veen W (2010) Understanding
lenging. Many aspects of the evolutionary history of this large polyspecificity of multidrug ABC transporters: Closing in on the gaps in ABCB1.
ubiquitous family are still to be unraveled. Better heterologous Trends in Biochemical Sciences 35: 36–42.
overexpression systems have to be developed to allow further Jones PM and George AM (2004) The ABC transporter structure and mechanism:
Perspective on recent research. Cellular and Molecular Life Sciences 61: 682–699.
biochemical and mechanistic studies. Additional atomic struc- Locher K (2009) Structure and mechanism of ATP-binding cassette transporters.
tures have to be produced to identify all conformation changes Philosophical Transactions of the Royal Society B Biological Sciences
during the ATP-driven transport cycle. The diverse mechanisms 364: 239–245.
 Algal Hydrogen Production
U M N Murthy and M L Ghirardi, Bioscience Center, National Renewable Energy Laboratory (NREL), Golden, CO, USA
ã 2013 Elsevier Inc. All rights reserved.

Glossary Photobioreactor A transparent reactor that allows growth

Catalytic H-cluster The catalytic core of the of cells under illumination; this term mostly refers to closed
[FeFe]-hydrogenase (HydA), termed the ‘H-cluster’, exists systems that have no direct exchange of gases and
as a [4Fe–4S] subcluster linked by a cysteine thiolate to contaminants with the environment, as opposed to open
a modified 2Fe subcluster with unique nonprotein ponds.
ligands. Radical SAM proteins A novel protein superfamily with
Fuel cells An electrochemical cell that converts a over 600 members that catalyze diverse reactions, including
source fuel into an electrical current. It generates unusual methylations, isomerization, sulfur insertion, ring
electricity inside a cell through reactions between a formation, anaerobic oxidation, and protein radical
fuel and an oxidant, triggered in the presence of an formation.
electrolyte.

Introduction Green Algal Pathways for H2 Production

Fossil fuels have been utilized as a source of energy for many The unicellular alga Chlamydomonas reinhardtii belongs to the
centuries, resulting in congestion, pollution, and growing group of eukaryotic green algae and lives in freshwater or
environmental stress. Global oil supplies are rapidly being in moist places. In addition to photosynthesis, unicellular
depleted and production levels are increasingly being reported green algae are able to perform many biotechnologically inter-
to be close to their peak. More importantly, zero CO2 emission esting metabolic reactions, such as fermentation and hydrogen
fuels are becoming increasingly important due to the con- photoproduction. The starting point for all biological solar-
straints of global climate change. Therefore, the development driven H2-production methods is the process of photosynthesis
of new systems to produce these fuels is one of the greatest (see Figure 1). Oxygenic photosynthesis involves a sequential
challenges facing our society. Solar energy is the most abun- chain of reactions that include light absorption, charge separa-
dant and accessible renewable energy source available for tion, electron transport, and dark CO2 fixation. Oxygenic
future sustainable production of fuel and electricity. For effec- organisms harness solar energy to extract electrons from H2O,
tive use of solar energy, it is important to develop more cost- which are required for CO2 fixation. Electrons generated from
effective systems with the improved ability to convert solar H2O molecules at photosystem II (PSII) are transferred to the
energy into chemical energy. In this context, hydrogen (H2) is cytochrome b6/f complex (b6f) and subsequently delivered to
considered to be a promising, clean energy carrier for future photosystem I (PSI). Through PSI, the electrons are transferred
technologies. The fact that the burning of H2 produces only to ferredoxin (Fd) (a 2Fe–2S protein). Normally, Fd shuttles
H2O increases its attractiveness, particularly in light of recent electrons to the enzyme ferredoxin–nicotinamide adenine
advances in hydrogen fuel cell technology. The direct photo- dinucleotide phosphate (NADP)-oxidoreductase (FNR) that
production of H2 by cyanobacteria and green algae is an alter- reduces NADPþ to NADPH, the direct source of reductant to
native to utilizing the reductant generated from photosynthetic convert CO2 into carbohydrates in the Calvin–Benson cycle.
water reduction for the fixation of CO2 into carbon-containing Concomitantly, protons (Hþ) are carried from the stromal
molecules. to the lumenal side of the thylakoids, creating a proton gradient
Hydrogen metabolism is primarily the domain of bacteria across the thylakoid membrane, which is utilized by the
and microalgae. Within these groups, it involves many taxo- enzyme adenosine triphosphate (ATP) synthase to generate
nomically diverse species, a variety of enzymes, and several the ATP that is also required for CO2 fixation. Under anaero-
metabolic pathways and processes. Green algae and cyanobac- bic conditions, reduced Fd efficiently binds to an [FeFe]-
teria are photoautotrophic organisms; they can grow under hydrogenase and donates electrons to its catalytic site, known
only sunlight and CO2, without organic sources of carbon. as the ‘H-cluster’ (HC). The function of the hydrogenase is to
It has been known for more than 60 years that, under anaero- combine protons (Hþ) and electrons (e) to form and release
bic conditions, unicellular green algae can metabolize H2, molecular H2. Photosynthetic reducing power can thus be parti-
either by uptaking it and using it as an electron donor in tioned between at least two pathways: CO2 reduction (under
CO2-fixation, or by evolving H2 in the light. Indeed, hydroge- aerobic conditions) and H2 production (under anaerobiosis). It
nase enzymes that either uptake or evolve H2 have been found is important to point out that CO2 reduction requires ATP,
in many green algae. This article briefly examines green algal whereas H2 photoproduction does not. Instead, the latter is
hydrogen metabolism and how it can be adapted for commer- downregulated in the absence of ATP production (see section
cial H2-production processes. H2 Production: Issues and Challenges). As expected, removal

66
 Bioenergetics | Algal Hydrogen Production 67

Oxidative
Starch CO2
respiration
NA(P)DH H2
Low light saturation O2-sensitivity
Chl Chl
antenna antenna
NADPH
FeFe
FNR NADP+
H2ase
H+
Competition for reductant
State transitions
e– Fd ATP
ADP + Pi
e– e–
PSII PQ pool PSI ATPase

H+ Nondissipated proton
H2O + gradient
O2 + H

Figure 1 Photosynthetic electron transport pathways in the green alga, Chlamydomonas reinhardtii. Photosynthesis extracts electron from H2O and
via the electron transport chain delivers them to the [FeFe]-hydrogenase where H2 is generated. The red circles denote the major barriers limiting
technological use of algae for photobiological H2 production. Adapted from Ghirardi ML and Mohanty P (2010) Oxygenic hydrogen
photoproduction – current status of the technology. Current Science 98: 499–507, with permission from Current Science.

of CO2 from the headspace enhances light-driven H2-evolution, the sulfur atom from a cysteine residue to a unique binuclear
indicating competition for electrons between the CO2-fixation iron–sulfur subcluster. Three other cysteines coordinate the
and the H2-evolution processes. [4Fe4S]-center to the protein in all known [FeFe]-hydrogenases.
Oxygenic H2 photoproduction results in the simultaneous Except for the bridging cysteine, the iron atoms of the 2Fe2S
generation of H2 and O2 in an 2:1 ratio as long as O2 is con- center are coordinated to carbon monoxide (CO) and cyanide
tinuously removed from the headspace. In the absence of active (CN) ligands, which are responsible for stabilizing the reduced
removal of O2, this mechanism can operate for only 30–90s form of the enzyme, and by a dithiomethylamine group linking
since O2 is a powerful inhibitor of the [FeFe]-hydrogenase reac- the two S atoms. One of the CO groups is found in a position
tion (and it is also a suppressor of [FeFe]-hydrogenase gene bridging both iron atoms when the enzyme is in an oxidized
transcription, which is discussed in detail in later sections). state, but its orientation has been proposed to change toward
An alternate source of electrons for H2 photoevolution is the distal Fe when the enzyme is in the reduced state. Highly
endogenous substrate degradation. This pathway is dependent conserved residues comprising several hydrophobic amino acids
on the reduction of the plastoquinone (PQ) pool by NADH are thought to be involved in the formation of H2 channels, thus
(released from the initial step of glycolysis), in a reaction connecting the catalytic site to the protein surface.
catalyzed by the PQ/NAD(P)H-oxidoreductase. Light absorp- In C. reinhardtii, a monomeric [FeFe]-hydrogenase of
tion by PSI and ensuing electron transport elevates the redox 48 kDa (named HYDA1) with high specific activity was first
potential of these electrons to the redox equivalent of ferre- isolated and biochemically characterized. More recent research
doxin, thus permitting the generation of molecular H2. revealed the presence and expression of a second [FeFe]-
hydrogenase (HYDA2) in C. reinhardtii that is 74% similar
and 68% identical to HYDA1. The transcripts for both hydro-
Enzymes for Algal Photohydrogen Production genases, HYDA1 and HYDA2, are selectively expressed upon
dark anaerobic incubation, suggesting regulation of gene
Hydrogenases are enzymes capable of producing or uptaking expression by O2. HYDA1, and most probably HYDA2, directly
molecular hydrogen. Hydrogenases are classified into three interact with reduced ferredoxin. The nucleus-encoded algal
main groups, according to the cofactor(s) they contain in hydrogenases are synthesized in the cytosol as precursor pro-
their catalytic site, as iron–iron [FeFe], nickel–iron [NiFe], or teins but the mature proteins are localized in the chloroplast
Fe-only hydrogenase. Algal hydrogenases belong to the class stroma. A transit peptide domain that routes the [FeFe]-
of [FeFe]-hydrogenases, they contain only Fe and S in their hydrogenases from the cytoplasm across the chloroplast enve-
catalytic site, and are typically involved in H2 production rather lope and into the chloroplast stroma has been identified at the
than H2 oxidation. They catalyze the reversible reduction of N-terminal region of the enzyme.
protons to H2:

2Hþ þ 2Fd ! H2 þ 2Fd

[FeFe]-Hydrogenases Are Sensitive to O2
[FeFe]-hydrogenases are small, monomeric enzymes of
45–49-kDa size containing a novel type of [Fe–S] cluster com- The metallo-cluster of the [FeFe]-hydrogenases is very sensitive
monly referred to as the ‘H-cluster’ at their catalytic center. to O2. This observation, together with the fact that hydrogenases
The HC comprises a conventional [4Fe–S] complex bridged by are located in the chloroplast, where PSII releases O2, causes H2
68 Bioenergetics | Algal Hydrogen Production

production rates to be usually low. In algal cells, O2 can segments, HYDE and HYDF, that are exclusively found in other
adversely affect enzymatic activity by deactivating previously organisms containing [FeFe]-hydrogenase but encoded as two
assembled [FeFe]-hydrogenases, and the isolated enzyme com- separate genes, HydE and HydF. The HydE proteins are highly
petes with CO for the same binding site in the HC of [FeFe]- homologous to the biotin synthase radical SAM proteins, and
hydrogenases. Oxygen inactivation is thought to occur by the HYDF shares homology with the large family of nucleoside
direct interaction of O2 with the [2Fe–2S] center on catalytic triphosphate (NTP)-binding proteins.
HC. In contrast to O2 inactivation, inhibition by CO is largely The specific role of HydE, HydF, and HydG proteins in
but not entirely reversible. The nature of the final oxygen [FeFe]-hydrogenase maturation is not clearly understood yet.
species bound to the inactivated enzyme is not known. How- Peters et al. proposed that the maturation proteins use com-
ever, it is known that diffusion of O2 gas is necessary for the mon amino acids or other metabolites to synthesize a pre-
inactivation of the enzyme, since the catalytic site of the cursor to the [FeFe]-hydrogenase active site, which is then
enzyme is embedded in the protein structure. transferred to the structural enzyme. HydE and HydG act on
Melis and his coworkers in 2000 demonstrated a novel HydF to assemble an active-site precursor containing all the
strategy to sustain green algal H2 production in the light. nonprotein ligands of the binuclear Fe subcluster, which is
The O2 sensitivity of H2 metabolism was bypassed in a two- subsequently transferred to the hydrogenase. It seems that
stage process, in which O2-evolution and H2-evolution reactions HydE is probably responsible for the synthesis of CO and
were temporarily separated. In the first stage, normal photo- CN ligands and HydG for the dithiomethylamine ligand.
synthesis, CO2-fixation, and release of O2 upon oxidation of The dithiomethylamine-bridged cluster is then transferred as
H2O enabled green algae to survive. In the second stage, the a whole to HydF to generate a complete, functional HC for
authors selectively and partially inactivated the O2-evolving insertion to the [FeFe]-hydrogenase apoprotein.
activity of PSII by incubating algal cultures in a sulfur-depleted
medium. Since respiratory O2 consumption is not affected
by sulfur deprivation, inactivation of O2 evolution facilitated The Interaction of [FeFe]-Hydrogenases with
the transition from the aerobic to an anaerobic state in a sealed Ferredoxin
photobioreactor. The establishment of anaerobiosis in the
photobioreactor induced the expression of the two [FeFe]- In green algae, H2 production couples to the photosynthetic
hydrogenases in algal cells and inhibited the expression of electron transport chain via ferredoxin. Ferredoxins are small
ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco). soluble [Fe–S] proteins which can be found in every phyloge-
This resulted in redirection of the flow of electrons from the netic group. They are mainly low-potential electron carriers
photosynthetic electron transport chain in the chloroplast involved in various metabolic pathways having diverse redox
away from carbon fixation and towards proton reduction. As partners. Ferredoxin distributes photosynthetic electrons not
a consequence, sulfur-deprived algae were able to produce H2 only to hydrogenase but also to other electron-dependent
for a total of 3–4 days. enzymes, the most important of which is ferredoxin-NAD(P)-
reductase (FNR) for CO2 fixation.
There are six ferredoxin genes in C. reinhardtii. The physio-
[FeFe]-Hydrogenase Maturation logical regulation and metabolic function of each of the six
ferredoxins has not yet been established, although it is assumed
Biosynthesis of an active [FeFe]-hydrogenase is a complex that both hydrogenases may associate with the photosynthetic
process. [FeFe]-hydrogenase maturation proteins were initially electron transfer ferredoxin (PetF, also called FDX1) which
discovered in the green alga C. reinhardtii. Posewitz et al. showed is involved in photosynthesis and it is a typical plant-type
that two novel radical S-adenosyl-methionine (radical SAM) [2Fe–2S] ferredoxin. Recently, Winkler et al. characterized the
proteins, HYDEF and HYDG, are required for C. reinhardtii kinetics of electron transfer processes between HydA1 and
[FeFe]-hydrogenase activity. Using a chemochromic sensor, PetF. Using an in vitro system by reconstituting part of the
a hydef mutant that was unable to produce hydrogen was photosynthetic electron transport chain consisting of plastocy-
isolated from a C. reinhardtii mutagenesis library. Moreover, anin, PSI, PetF, and [FeFe]-hydrogenase, they demonstrated
the HYDEF gene was found to be adjacent to the HYDG that residue K396 of HydA1 is crucial for a successful binding
gene, and both were shown to be present in all other [FeFe]- and electron transfer between PetF and HydA1. However, other
hydrogenase-containing organisms, although their role was studies by Terauchi et al. found that each of the six FDX genes is
unknown at that time. In the absence of a functional HYDEF, differentially regulated in response to changes in nutrient sup-
both hydrogenase structural genes were induced but no ply and/or anoxia. In this regard, more detailed understanding
hydrogenase activity was detected in the mutant. Complemen- of the hydrogenase–ferredoxin association mechanism and
tation of the mutant with genomic DNA containing a func- kinetics is necessary for engineering hydrogenase to divert a
tional copy of the HYDEF gene restored hydrogenase activity in greater electron flux to the H2 production pathway and thus to
the algae. Later on, King et al. showed that the heterologous optimize photobiological H2 production.
expression of active algal [FeFe]-hydrogenase in the bacterium,
Escherichia coli, could be accomplished by co-transforming the
HYDA1 structural gene along with both the HYDEF and HYDG H2 Production: Issues and Challenges
genes, and that all these genes were required and sufficient for
the expression of a functional [FeFe]-hydrogenase in this bac- Photobiological H2 production by C. reinhardtii has become an
terium. The algal HYDEF gene encodes for two unique protein important research field because of its potential to be applied
 Bioenergetics | Algal Hydrogen Production 69

in renewable energy production. To realize the economic

potential of producing hydrogen photobiologically, several
challenges must be overcome. Foremost, cellular metabolism
and the basic biochemistry supporting this process must be
understood. Following are the major issues and challenges that
need to be addressed.

O2 Sensitivity
The simultaneous photoproduction of H2 and O2 from H2O
in green alga depends on O2 tolerance of the reversible
hydrogenase enzyme. When oxygenic photosynthesis is active,
and unless strict measures are taken to remove O2, green algal
H2-production activity is transient owing to the rapid inactiva-
tion of the reversible hydrogenase by photosynthetic O2. Sus-
tained hydrogen production will only be possible when the
hydrogenase enzyme remains active under O2-evolving condi-
tions. Different strategies for development of O2 tolerant
hydrogenases have included molecular engineering of the Figure 2 Photograph of the photobioreactor used for H2 production
hydrogenases by either random or site-directed mutagenesis from Chlamydomonas reinhardtii culture showing probes used for
to remove O2 sensitivity. The latter has mostly focused on monitoring and the controlling the system. Hydrogen bubbles
physical restriction of O2 access catalytic site by reducing the emanate toward the surface of the culture and are collected in a
O2 diffusion rate or shielding the active catalytic site. Although liquid-accumulating bottle (not shown).
the results are encouraging from a scientific perspective in
understanding enzyme structure and function, none of these
approaches has yielded biotechnologically significant hydrogen- immobilization of sulfur/phosphorous-deprived algae in thin
producing strains yet. Ca2þ-alginate film matrices has extended H2 photoproduction
An alternative approach to sustain H2 production in the for more than 150 h and also increased the stability of H2
light is to use sulfur deprivation to partially inactive O2 evolu- photoproduction activity in the presence of atmospheric O2.
tion, as discussed above. The novel application of this process This work shows potential for further significant improve-
has led to significant and sustainable light-dependent release ments in both the rate and efficiency of H2 production under
of H2 by algal cultures, although at low-conversion efficiencies sulfur-deprived conditions.
due to limited PSII activity. Several approaches have been
examined to increase the yield of hydrogen in C. reinhardtii
Reduced Antenna Size
under sulfur-deprived condition, including optimization of the
light and pH conditions in the photobioreactors optimization Photosynthesis and H2 production in unicellular green algae
of the medium composition and synchronization of cell divi- can in principle operate with a nearly 100% photon utiliza-
sion (see Figure 2). Kruse et al. reported a significant increase tion efficiency, making them potentially efficient biocatalysts
in the rate and duration of H2 photoproduction in sulfur- for the generation of H2 from sunlight and water. However,
deprived mutants that are starch over-accumulators and green algal cultures show poor light utilization efficiency under
blocked in state transitions. Another advance related to the direct sun light and can waste up to 80% of the absorbed
sulfur-deprived process came with the recent discovery of a irradiance. The reason for this is that green algal photosynthe-
mutant affected in sulfate permease activity, which is required sis normally saturates at about one-fifth of full sunlight inten-
for transport of sulfate into the chloroplast. This mutant is a sity or less. This optical property of the cells would further
potential candidate for H2 photoproduction without the need lower the productivity of a commercial hydrogen production.
to totally remove sulfate from the growth medium. Enhanced hydrogen production may be achieved by engineer-
The reported rates of H2 production by sulfur-deprived ing the antenna size to suppress fluorescence and heat dissipa-
cultures is still far below the maximum potential rate of H2 tion that causes a reduction on antenna efficiency.
photoproduction for an algal system, mainly due to partial
inactivation of PSII. Furthermore, the system is difficult to
Competition for Reductants
scale up and maintain. These challenges, which are related
partly to the photobioreactor design, might be addressed by Competition from the CO2-fixation pathway for electrons
immobilizing the green algae. The immobilization technique from H2O is a major concern for sustained H2 production,
effectively separates cells from the liquid phase, significantly as discussed earlier in the article. This concern is based on the
increases the cell density and, as a consequence, allows for higher affinity of ferredoxin for FNR than hydrogenase, which
more efficient light utilization on a per area basis. Laurinavi- could result in substantial drainage of electrons away from H2
chene, et al. in 2006 demonstrated that sulfur-deprived, immo- production. Therefore, improved H2 production depends on
bilized cells of a nonmotile mutant of C. reinhardtii on glass interaction of [FeFe]-hydrogenases with the electron-carrying
fibers significantly increase the duration of H2 production. protein ferredoxin. Agapakis et al. fused the hydrogenase
More recently, Kosourov and Seibert, demonstrated that from C. acetobutylicum to ferredoxin from spinach or from
70 Bioenergetics | Algal Hydrogen Production

C. acetobutylicum using flexible protein linkers of various the molecular regulation of the genes encoding hydrogenases
lengths and showed a threefold increase in H2 production by and accessory proteins needed for assembly of hydrogenases,
a nonphotosynthetic, recombinant E. coli system. Moreover, as well as in identifying and addressing the major barriers to a
chimeras between ferredoxin and various ferredoxin reductases future commercial process. With increasing interest from the
have been shown to be functional in vitro, with improved scientific community and the public in clean and renewable
electron transfer rates, presumably due to the increased local energy sources, rapid progress could bring this technology
concentration of reduced ferredoxin. This approach may pro- to fruition in the not-so-long future. Moreover, the possibilities
vide new targets for metabolic engineering of H2 production in and challenges within synthetic biology should be further
the future. explored for creating green algae with a high potential for H2
production.

Downregulation of Electron Transport by the Proton Gradient

See also: Bioenergetics: Ferredoxin; Hydrogenases, Structure and
Under sulfur-deprived conditions, very little CO2 is fixed into
Function; Renewable Hydrogen from Biomass.
glucose, mainly because of the decrease in Rubisco levels.
These sulfur-deprived, anaerobic cultures use very little ATP,
as a consequence, the proton gradient generated by photo-
synthetic electron transport is not dissipated. This causes a Further Reading
decrease in the rate of electron transport as demonstrated by
Agapakis CM, Ducat DC, Boyle PM, et al. (2010) Insulation of a synthetic hydrogen
the acceleration in the rate of H2 production observed upon
metabolism circuit in bacteria. Journal of Biological Engineering 4: 3.
addition of uncouplers. Although, mutants defective in one or Ghirardi ML (2006) Hydrogen production by photosynthetic green algae. Indian Journal
other ATPase subunits have been generated, their uncoupling of Biochemistry and Biophysics 43: 201–210.
properties are yet to be studied. These mutants may be able to Ghirardi ML, Dubini A, Yu JP, and Maness PC (2009) Photobiological
circumvent the cellular regulatory process that downregulate hydrogen-producing systems. Chemical Society Reviews 38: 52–61.
Ghirardi ML and Mohanty P (2010) Oxygenic hydrogen photoproduction – current
PSII activity at the level of the plastoquinone pool and limit H2 status of the technology. Current Science 98: 499–507.
production. Ghirardi ML, Posewitz MC, Maness PC, et al. (2007) Hydrogenases and hydrogen
photoproduction in oxygenic photosynthetic organisms. Annual Review of Plant
Biology 58: 71–91.
State Transition Ghirardi ML, Zhang JP, Lee JW, et al. (2000) Microalgae: A green source of renewable
H-2. Trends in Biotechnology 18: 506–511.
Oxygenic photosynthetic organisms adapt to varying energetic King PW, Posewitz MC, Ghirardi ML, and Seibert M (2006) Functional studies of [FeFe]
requirements by transitioning between linear and cyclic elec- hydrogenase maturation in an Escherichia coli biosynthetic system. Journal of
Bacteriology 188: 2163–2172.
tron transport, through the so-called state transitions. If the Kruse O, Rupprecht J, Bader KP, et al. (2005) Improved photobiological H-2 production
ATP requirement is higher than what is provided by linear in engineered green algal cells. Journal of Biological Chemistry 280: 34170–34177.
electron transport alone, the cultures transition to state 2, Laurinavichene TV, Fedorov AS, Ghirardi ML, Seibert M, and Tsygankov AA (2006)
where cyclic electron transport predominates. Anaerobiosis Demonstration of sustained hydrogen photoproduction by immobilized,
sulfur-deprived Chlamydomonas reinhardtii cells. International Journal of
leads to the establishment of state 2 and, as a result, additional
Hydrogen Energy 31: 659–667.
limitations are placed on the rates of H2 photoproduction. Melis A and Happe T (2001) Hydrogen production. Green algae as a source of energy.
The relevance of the state 2 limitation on H2 photoproduction Plant Physiology 127: 740–748.
by sulfur-deprived culture was recently demonstrated by Kruse Melis A, Seibert M, and Ghirardi ML (2007) Hydrogen fuel production by transgenic
et al., who used a chlorophyll fluorescence video-imaging microalgae. Transgenic Microalgae as Green Cell Factories 616: 108–121.
Melis A, Zhang LP, Forestier M, Ghirardi ML, and Seibert M (2000) Sustained
screening procedure to identify mutants of C. reinhardtii photobiological hydrogen gas production upon reversible inactivation of oxygen
blocked in state 1. They isolated one mutant, stm6, with rates evolution in the green alga Chlamydomonas reinhardtii. Plant Physiology
of H2 photoproduction 13 times higher than its wild-type 122: 127–135.
strain, and with extended H2 photoproduction activity under Mus F, Dubini A, Seibert M, Posewitz MC, and Grossman AR (2007) Anaerobic
acclimation in Chlamydomonas reinhardtii – anoxic gene expression, hydrogenase
sulfur deprivation. These results underscore the importance of
induction, and metabolic pathways. Journal of Biological Chemistry
molecular engineering to address this particular limitation to 282: 25475–25486.
algal H2 production technologies. Peters JW, Szilagyi RK, Naumov A, and Douglas T (2006) A radical solution for the
biosynthesis of the H-cluster of hydrogenase. FEBS Letters 580: 363–367.
Posewitz MC, King PW, Smolinski SL, et al. (2004) Discovery of two novel radical
S-adenosylmethionine proteins required for the assembly of an active [Fe]
Conclusion hydrogenase. Journal of Biological Chemistry 279: 25711–25720.
Prince RC and Kheshgi HS (2005) The photobiological production of hydrogen:
Biological hydrogen production is not only a challenging but Potential efficiency and effectiveness as a renewable fuel. Critical Reviews in
Microbiology 31: 19–31.
also promising biotechnology area, with the potential to help Stripp ST and Happe T (2009) How algae produce hydrogen–news from the
solve environmental and energy-related problems. The applica- photosynthetic hydrogenase. Dalton Transactions 45: 9960–9969.
tion of biological H2 production as a mature technology, how- Terauchi AM, Lu SF, Zaffagnini M, et al. (2009) Pattern of expression and substrate
ever, depends on research advances to address improvements in specificity of chloroplast ferredoxins from Chlamydomonas reinhardtii. Journal of
Biological Chemistry 284: 25867–25878.
H2-production rates and efficiency through a combination of
Winkler M, Kuhlgert S, Hippler M, and Happe T (2009) Characterization of the key
genetically engineering microorganisms and developing bior- step for light-driven hydrogen evolution in green algae. Journal of Biological
eactors. Significant progress is being made in understanding Chemistry 284: 36620–36627.
 Amine Oxidases
G Floris, University of Cagliari, Monserrato (CA), Italy
A Finazzi Agrò, University of Rome, Rome, Italy
ã 2013 Elsevier Inc. All rights reserved.

Glossary (e.g., spermine and sperimidine) strongly interacting

Amines Hydrocarbon compounds bearing an amine group. with nucleic acids involved in many important cellular
They are called primary, secondary, or tertiary when functions.
nitrogen binds two, one, or zero hydrogen atoms. Primary amines Metabolically derived from amino
Oxidases Enzymes that oxidize substrates using acids by decarboxylation. Primary amines often
molecular oxygen. show potent pharmacological or hormonal activity
Polyamines Hydrocarbon compounds bearing (e.g., histamine, serotonin, GABA, and
both primary and secondary amino groups noradrenaline).

Classification corresponding aldehyde and ammonia, and FADH2 is oxidized

back to FAD by oxygen with the formation of hydrogen perox-
The oxidative deamination of mono-, di-, and polyamines is ide. MAO A oxidizes dopamine, noradrenaline, and serotonin
catalyzed by a number of enzymes called ‘amine oxidases’ whereas MAO B preferentially oxidizes benzylamine and phen-
(AOs) that exhibit different patterns of substrate specificity ylethylamine. Adrenaline, kynuramine, tyramine, and trypt-
and inhibitor sensitivity and also differ in their action mecha- amine are substrates for both enzymes.
nism. AOs are divided into two main categories, depending on MAO A and B have been implicated in apoptosis, immuno-
the nature of the cofactor involved. Monoamine oxidases suppression, cytotoxicity, cell growth, and proliferation. These
(MAOs) and polyamine oxidases (PAOs) belong to the first enzymes play a protective role in the body by preventing the
class and utilize flavin adenine dinucleotide (FAD) as the entry of amines at renal and intestinal level or by oxidizing
redox cofactor (Figure 1(a)). The second class is represented them in blood. Liver MAOs are important for the control of
by enzymes having a tightly bound CuII ion and a carbonyl-type blood pressure by regulating the amines level. For instance,
moiety identified as 6-hydroxydopa quinone (2,4,5-trihydrox- MAO A can oxidize circulating serotonin thus preventing its
yphenethylamine quinone; TPQ or TOPA) at the active site effects on heart and vascular system. Both enzymes have
(Figure 1(b)). important roles in the metabolism of neurotransmitters and
other biogenic amines in the brain and are implicated in a large
number of neurological and psychiatric disorders. As a matter
Distribution, Reaction Mechanism, and of fact, MAO A inhibitors are used as antidepressants whereas
Physiological Roles MAO B inhibitors have an important role in the treatment of
Parkinson’s disease.
Monoamine Oxidases
A further important role of MAOs is recently emerging, that
Two forms of MAOs (MAOs, amine: oxygen oxidoreductase is, their role in the demethylation of histone lysines and argi-
(deaminating, flavin-containing); EC 1.4.3.4) have been de- nines, an essential process in the regulation of gene expression
scribed, namely MAO A and MAO B, the former made by 527 and genomic imprinting.
amino acid residues and the latter by 520, with an overall 70%
identity. Both enzymes have a FAD covalently bound through
Polyamine Oxidases
its C8a-position to a cysteine (Cys406 in MAO A and Cys397 in
MAO B) side chain (Figure 1(a)). Both forms of MAOs have PAOs (N1-acetylspermidine:oxidoreductase (deaminating); EC
been crystallized from many different sources. 1.5.3.11) have been classified as part of flavoprotein superfam-
MAOs are ubiquitous in cells of most mammalian species, ily but in this case FAD is noncovalently bound to the protein.
the notable exception being erythrocytes, and are tightly asso- PAOs are monomeric enzymes with a molecular mass ranging
ciated with the mitochondrial outer membrane. The distribu- from 53 kDa to 63 kDa. PAOs are found in vertebrates
tion of MAOs has been mainly studied in brain: MAO A is and plants. Enzymes with characteristics similar to those of
found in catecholaminergic neurons, whereas MAO B is abun- vertebrates’ enzymes are present in yeasts and amoebae,
dant in serotonergic and histaminergic neurons and glial cells. whereas bacteria and protozoans contain enzymes resembling
Some tissues mainly contain MAO A (human placenta and those of plants. Plant PAOs have been isolated and character-
bovine tyroid), while other tissues contain predominantly ized, particularly from the Gramineae oat, maize, barley,
MAO B (human platelets and bovine liver and kidney). wheat, and rye. The enzyme from maize seedlings has been
MAOs oxidize primary amino groups of arylalkyl amines to crystallized.
form an imine intermediate with the concomitant reduction Much alike the catalytic mechanism of MAOs, the oxidation
of flavin to FADH2. The imine is then hydrolyzed to the of polyamines can be divided into two half-reactions: (1) flavin

87
88 Bioenergetics | Amine Oxidases

reduction upon polyamine oxidation followed by (2) flavin PAOs preferentially oxidize acetyl spermine and acetyl spermi-
reoxidation by molecular oxygen. PAOs catalyze the oxidation dine: spermidine and putrescine are the respective reaction pro-
of polyamines’ secondary amino groups yielding different pro- ducts, together with 3-aminopropanal and hydrogen peroxide.
ducts depending on the organism considered. Mammalian Oxidation of spermine by plant PAOs gives 1,3-diaminopro-
pane, hydrogen peroxide, and 1-(3-aminopropyl)-4-aminobu-
tanal. The latter spontaneously cyclizes to 1-(3-aminopropyl)
MAO O pyrrolinium that undergoes further spontaneous rearrange-
ments to 1,5-diazobicyclo[4.3.0.]nonane (Scheme 1). The oxi-
NH CH C
S R dation of spermidine by plant PAOs gives 1,3-diaminopropane,
CH2 hydrogen peroxide, and 4-aminobutanal that yields 1-pyrroline
H2C N N O
O by spontaneous cyclization. At variance with MAOs and copper/
topaquinone (TPQ) AOs, the oxidation of polyamines by PAOs
N
H3C N H does not release ammonia.
O PAOs play an important role in the regulation of intracellu-
O
(b) O lar polyamines level, and seem to be important for homeosta-
(a)
sis and cell survival.
O
NH CH C Copper Amine Oxidases
H CH2 Copper amine oxidases (amine: oxygen oxidoreductase (dea-
CH2 N minating; copper containing); EC 1.4.3.6) (CAOs) are homo-
CH2 dimers in which each subunit (Mw 70–90 kDa) contains a
tightly bound type II copper ion and a TPQ. TPQ is derived
CH2 O
from a posttranslational modification of a tyrosyl residue,
CH2 O found in a strictly conserved amino acid consensus sequence
NH CH asn-tyr-asp/glu (NYD/E). The pink color of CAOs stems from
an intense absorption band around 500 nm due to the oxi-
C O
dized TPQ. CAOs catalyze the oxidative deamination of pri-
(c)
mary amino groups of mono-, di-, and polyamines, abstracting
Figure 1 Structure of the (a) FAD covalently bound in MAOs; (b) the two electrons from amines and transferring them to molecular
oxidized form of TPQ; and (c) lysyl tyrosylquinone. oxygen, to form the corresponding aldehyde, ammonia, and

(P)
(1)
+ O2, H2O
H2N (CH2)3 NH (CH2)4 NH2 H2N (CH2)3 NH2 + HOC(CH2)3 NH2
− H2O2

+H2O −H2O

N
(P)
(a)
(2)
+ O2, H2O
H2N (CH2)3 NH (CH2)4 NH (CH2)3 NH2 H2N (CH2)3 NH2 + HOC (CH2)3 NH (CH2)3 NH2
−H2O2

+H2O −H2O

H +H+
N N NH2 (CH2)3 N
−H+
(c) (b)
(M)
(3)
+ O2, H2O
H2N (CH2)3 NH (CH2)4 NH2 H2N (CH2)2 CHO + H2N (CH2)4 NH2
−H2O2

(M)
(4)
+ O2, H2O
H2N (CH2)3 NH (CH2)4 NH (CH2)3 NH2 H2N (CH2)2 CHO + NH2 (CH2)4 NH (CH2)3 NH2
−H2O2

Scheme 1 Spermidine and spermine oxidation catalyzed by plant (P) and mammalian (M) PAOs. Spermidine (1, 3); Spermine (2, 4); (a) 1-pyrroline;
(b) 1-(3-aminopropyl)-pyrrolinium; and (c) 1,5-diazobicyclo[4.3.0.]nonane. Arrows indicate the cleavage of polyamines.
 Bioenergetics | Amine Oxidases 89

hydrogen peroxide. Again, the catalytic mechanism can be The enzyme from bovine plasma has been crystallized.
divided into two half-reactions: (1) enzyme reduction at the Tissue-bound AOs are often indicated somewhat mislead-
quinone moiety (TPQ ! TPQH2) by substrate followed by (2) ingly as semicarbazide-sensitive amine oxidases (SSAOs),
reoxidation by molecular oxygen since all CAOs, at variance with FAD-dependent enzymes,
are inhibited by semicarbazide and other carbonyl reagents.
Eox þ R  CH2  NHþ þ
3 ! Ered  NH3 þ R  CHO [1] Intracellular SSAOs are widely distributed: smooth muscle
cells of vascular tissue of all mammalian species are good
Ered  NHþ þ
3 þ O2 þ H2 O ! Eox þ NH4 þ H2 O2 [2]
sources of these enzymes, which have been also detected in
uterus, ureter and vas deferens, in ox dental pulp, in human
The group of CAOs includes the following:
umbilical artery, in rat adipocytes, and chondrocytes.
1. Diamine oxidases (DAOs), ubiquitous enzymes occurring Plasma AOs may be considered key enzymes in cell
in prokaryotes as well as eukaryotes (bacteria, fungi, plants, growth and differentiation processes and, thus, in tumor
and animals). Several DAOs have been crystallized from development and growth regulation. Moreover, the
bacteria, from the yeast Hansenula polymorpha, and from increased BzAO activity in blood serum of pregnant
pea seedlings. Plant DAOs from various species have been women supports the possibility that this enzyme has a
purified to homogeneity and characterized, the best known protective role against the polyamines released from the
and studied being those from lentil (Lens esculenta) and pea fetoplacental unit. SSAOs seem to play an important role
(Pisum sativum) and from latex of the shrub Euphorbia char- in vascular wall. Moreover, these enzymes might be
acias. In mammals, the best-known enzymes are those from involved in the regulation of glucose metabolism (perhaps
pig kidney and intestine and human placenta. DAOs are via the H2O2 produced) and in the regulation of leukocyte
mainly active on short aliphatic diamines, such as putrescine trafficking in endothelial cells. SSAOs have also been shown
(1,4 diaminobutane) and cadaverine (1,5 diaminopentane). to be a new class of DNA-binding proteins: in the presence of
The physiological role of DAOs is multifaceted, because polyamines, they bind DNA and oxidize DNA-bound poly-
this class includes several enzymes with different localiza- amines. A structural similarity between SSAOs and VAP-1, a
tions and substrates. Bacteria and yeasts can utilize amines protein involved in cellular adhesion, has been observed and
as nitrogen and carbon sources through the reaction with a human SSAO/VAP-1 protein has been crystallized.
AO. Plant DAOs are implicated in wound healing, in detox- 3. The mammalian extracellular matrix-bound lysyl oxidase
ification, and in cell growth by regulating the intracellular [EC 1.4.3.13] (LOX), a 30-kDa protein, differs from other
di- and polyamine levels, and the aldehyde products might members of the group because a lysyl tyrosyl quinone
have a key role in the biosynthesis of some alkaloids. The (LTQ) rather than TPQ is the redox cofactor (Figure 1(c)).
function of DAOs in mammals is even more diverse and LTQ is formed by oxidation of a conserved tyrosine residue
elusive and a plethora of physiological functions, some- and subsequent intramolecular cross-linking with the
times opposite, have been described. AO activity is found e-amino group of a conserved lysine. LOX catalyzes the
in many tissues, the highest levels being in decidual cells of oxidative deamination of lysyl residues in elastin and col-
placenta, in kidney tubular epithelial cells, and in intestinal lagen allowing the formation of cross-links essential for the
epithelial cells. These localizations may suggest a barrier structure of these proteins. The genetic or acquired decrease
function for DAOs to prevent the entry of diamines and of LOX activity is accompanied by severe pathological con-
polyamines in the body. Furthermore, these enzymes may ditions such as tendon laxity and formation of aneurisms
keep at bay the endogenous histamine, which otherwise in arteries.
can cause several pathological conditions such as allergy, A LOX from Pichia pastoris has been crystallized and
peptic ulcer, and anaphylactic reactions. Several observa- characterized. This enzyme, cloned and overexpressed,
tions point to a relationship between AO activity and shows TPQ as the redox cofactor. Pichia pastoris LOX dis-
growth, both in normal and tumor tissues, that is, in cell plays catalytic activity toward tropoelastin (the un-cross-
proliferation and differentiation. DAOs have also been pro- linked form of elastin), and lysine and lysine-containing
posed as immune response modulators. It has been demon- peptides are the preferred substrates for this enzyme. In this
strated that human placental DAO is identical to the respect, it resembles the LTQ-containing mammalian AOs.
amiloride-binding protein and thus in some way involved
in the regulation of epithelial ion transport. In analogy with
MAOs, also DAOs have been proposed to be involved in
demethylation of histones, in particular of methylated argi- Concluding Remarks
nines. In this case, their importance might be not only the
modification of histones but also the production of hydro- In the past few years, evidence has accumulated about the
gen peroxide just at nuclear level. physiological relevance of hydrogen peroxide, which is gener-
2. The mammalian extracellular soluble and intracellular ated in the catabolism of mono-, di-, and polyamines by all
tissue-bound AOs are able to metabolize mono-, di-, and AOs. Hydrogen peroxide, which is always formed in the reac-
polyamines, even though in vitro they are preferentially active tions catalyzed by AOs, is more and more considered either a
toward nonphysiological amines such as benzylamine. Extra- crucial reagent for important biochemical processes or a signal
cellular AOs are generally called either plasma and serum and a defense molecule, rather than a noxious waste product.
AOs or benzylamine oxidases (BzAOs). The better-known This reactive oxygen species is toxic at high concentration.
BzAOs are those from bovine, swine, and equine plasma. However, at lower concentrations it regulates the cell number
90 Bioenergetics | Amine Oxidases

during embryonic development as well as the proliferation and Ciccone DN, Su H, Hevi S, et al. (2009) KDM1B is a histone H3K4
adhesive properties of endothelial and smooth muscle cells. demethylase required to establish maternal genomic imprints. Nature 461:
415–419.
Hydrogen peroxide appears to be involved in the impairment
Dandrifosse G (ed.) (2009) Biological Aspects of Biogenic Amines, Polyamines and
of cell growth and proliferation, in the regulation of many Conjugates. Trivandrum, India: Transworld Research Network.
genes and transcription factors, and in the transduction of De Colibus L, Li M, Binda C, et al. (2005) Three-dimensional structure of human
cellular signals in many living species. In plants, hydrogen monoamine oxidase A (MAO A): Relation to the structures of rat MAO A and human
peroxide might be utilized by peroxidases in basic physiologi- MAO B. Proceedings of the National Academy of Sciences of the United States
of America 102: 12684–12689.
cal events, such as development, polymerization of lignin and Duff AP, Cohen AE, Ellis PJ, et al. (2003) The crystal structure of Pichia pastoris lysyl
suberin precursors, and catabolism of indoleacetic acid, in oxidase. Biochemistry 42: 15148–15157.
response to wounding or to pathogen invasion. Floris G and Mondovı̀ B (eds.) (2009) Copper Amine Oxidases. Boca Raton, London
NY: CRC Press, Taylor & Francis Group.
Jalkanen S and Salmi M (2001) Cell surface monoamine oxidases: Enzymes in
search of a function. New EMBO member’s review. The EMBO Journal 20:
See also: Bioenergetics: Quinones. 3893–3901.
Janes SM, Mu D, Wemmer D, et al. (1990) A new redox cofactor in eukaryotic enzymes:
6-hydroxydopa at the active site of bovine serum amine oxidase. Science
248: 981–987.
Further Reading Mure M, Mills SA, and Klinman JP (2002) Current topics. Catalytic mechanism
of the Topa quinone containing copper amine oxidases. Biochemistry
Binda C, Coda A, Angelini R, et al. (1998) Crystallization and preliminary X-ray analysis 41: 9269–9278.
of polyamine oxidase from Zea mays L. Acta Crystallographica Section D Shi Y, Lan F, Matson C, et al. (2004) Histone demethylation mediated by the nuclear
54: 1429–1431. amine oxidase homolog LSD1. Cell 119: 941–953.
 Anaplerosis
R R Russell III, Yale University School of Medicine, New Haven, CT, USA
H Taegtmeyer, The University of Texas Medical School at Houston, Houston, TX, USA
ã 2013 Elsevier Inc. All rights reserved.

Glossary b-Oxidation The oxidative metabolism of fatty

Anaplerosis The entry of substrates into the citric acid cycle acids through a cycle of reactions that removes
as intermediates other than acetyl-CoA, thereby increasing successive two-carbon units (acetyl-CoA) from the
the citric acid cycle pool size. fatty acid.
Carboxylation The introduction of a carboxyl group into a
compound.

The word anaplerosis (from the Greek, meaning to fill up) was metabolic homeostasis through the regulation of insulin secre-
coined in the 1960s by Sir Hans Kornberg to describe pathways tion from the b-islet cells of the pancreas. We now focus on the
that replenish intermediates in metabolic cycles. A metabolic mitochondrial anaplerotic pathways.
cycle transforms a substrate with little or no change in the
concentration of the cycle’s constituents, the central process
in energy substrate metabolism. A classic example for a meta- Anaplerotic Pathways
bolic cycle is the citric acid cycle. Here anaplerotic pathways
Substrates for Anaplerosis
mediate the entry of metabolites into the cycle as compounds
other than acetyl-CoA and thereby replenish the pool size of The known anaplerotic pathways for the citric acid cycle are
the citric acid cycle. Other anaplerotic pathways are present in depicted in the schematic drawing of Figure 2. As a key com-
eukaryotic and prokaryotic organisms; however, this article ponent of all living cells, the citric acid cycle is highly organized
focuses on anaplerosis as it relates to the citric acid cycle in and responds instantly to changes in its environment. The
mammalian tissues and the importance of anaplerosis in the citric acid cycle is tied into a distributive system of energy
maintenance of organ function. transformation reactions (Figure 1) and flux through the ana-
Cycles have evolved because in a competitive environment plerotic pathways. The primary substrates for anaplerosis are
the chances for survival are greatest if resources are used in an lactate and alanine as precursors of pyruvate, although gluta-
optimal way. For example, energy transfer in heart muscle is mate can be a physiologically significant anaplerotic substrate
linked to a series of cycles, beginning with the circulation and as well. In addition, the branched chain amino acids valine and
ending with actin–myosin cross-bridge formation (Figure 1). isoleucine, as well as odd-chain fatty acids and their metabolic
Steady-state concentrations of metabolic intermediates in a end product, propionate, serve as anaplerotic substrates.
cycle depend on their rates of synthesis and degradation. In
other words, substrates entering the citric acid cycle as citrate
Entry at Malate and Oxaloacetate
(via the condensation of acetyl-CoA with oxaloacetate) do not
cause a net change in the citric acid cycle pool size because the two In the heart the major anaplerotic substrate in the heart is
carbons of acetyl-CoA are ultimately lost as CO2 in the isocitrate pyruvate. Although the majority of pyruvate entering the mito-
and the a-ketoglutarate dehydrogenase reactions, respectively. In chondria is oxidatively decarboxylated (and most likely by CO2
contrast, anaplerosis supplies compounds to the citric acid cycle arising from pyruvate decarboxylation) and enters the citric acid
through reactions other than those catalyzed by citrate synthase. cycle as acetyl-CoA, a portion is also carboxylated and enters the
These compounds replenish carbon intermediates lost primarily four-carbon (C-4) pool of the citric acid cycle. This conversion is
as amino acids (glutamate or aspartate) or as oxaloacetate. catalyzed by either pyruvate carboxylase (PC, E.C. 6.4.1.1),
The role of anaplerosis in the citric acid cycle is threefold. forming oxaloacetate or by nicotinamide adenine dinucleotide
First, as intermediates are drained away during biosynthetic phosphate (NADP)-dependent malic enzyme (E.C. 1.1.1.40),
processes such as gluconeogenesis, glyceroneogenesis, and forming malate. While the NADP-dependent malic enzyme
amino acid synthesis, anaplerotic pathways replace them, reaction is reversible, the reaction in the direction of malate
assuring sufficient carbon flux for citrate synthesis and, production is slow and unlikely to play a significant role in
hence, allow citric acid cycle flux to proceed unimpaired. anaplerosis. Another provider of C-4 intermediates in the citric
These aspects of anaplerosis are especially important in the acid cycle is aspartate, which undergoes transamination with
liver and renal cortex. Second, anaplerosis facilitates energy a-ketoglutarate via aspartic aminotransferase to form oxaloace-
transfer in organs with high rates of energy turnover, such as tate and glutamate. In contrast to the carboxylation of pyruvate,
the heart and skeletal muscle, especially under situations such the entry of aspartate as oxaloacetate is balanced by the loss of
as ischemia in which normal functioning of the citric acid cycle a-ketoglutarate from the citric acid cycle, resulting in the homeo-
is impaired. Third, anaplerosis also regulates whole-body stasis of citric acid cycle intermediates.

105
106 Bioenergetics | Anaplerosis

Entry via a-Ketoglutarate is present in a variety of tissues, including the liver, kidney,
brain, heart, and skeletal muscle.
Mitochondrial a-ketoglutarate can be readily formed from
glutamate via transamination with pyuruvate (forming ala-
nine) or oxaloacetate (forming aspartate). The transamina- Entry via Succinyl-CoA
tion of glutatmate with oxaloacetate by glutamate
The citric acid cycle intermediate succinyl-CoA plays an impor-
oxaloacetate transaminase (E.C. 6.2.1.1) results in a gain of
tant role in fatty acid and amino acid metabolism because it is
a C-5 intermidate at the same time that a C-4 intermediate is
the entry point of odd-chain fatty acids, propionate, and the
lost from the citric acid cycle. Of importance is that the
branched chain amino acids valine and isoleucine into the
mitochondrial pools of a-ketoglutarate and glutamate are in
citric acid cycle. Substrate entry as a-ketoglutarate or succinyl-
equilibrium and therefore labeling patterns of glutamate are
CoA, in contrast to other anaplerotic pathways, is associated
identical to those for a-ketoglutarate. As discussed below, this
with the generation of energy-rich phosphates via a substrate-
relationship between a-ketoglutarate and glutamate can be
level phosphorylation. The reaction catalyzed by succinyl-CoA
exploited in measuring citric acid cycle flux, relative rates of
synthetase (E.C. 6.2.1.4) normally favors the conversion of
contribution of substrates to the citric acid cycle, and rates
succinyl-CoA to succinate and leads to substrate level phos-
of anaplerosis. Furthermore, glutamate can be converted to
phorylation of GDP to GTP. This energy-providing pathway
a-ketoglutarate and NHþ 4 by glutamate dehydrogenase, which becomes important under anaerobic conditions when ATP
generation by oxidative phosphorylation is inhibited, such as
in the setting of ischemia.
Ca2+
Circulation
Krebs NADH H+ Entry via Fumarate
cycle
H+ PCr
In addition to the amino acids phenylalanine and tyrosine
entering as fumarate, the purine nucleotide cycle enriches the
Ca2+
ATP citric acid cycle based on the net reaction
Ca2+
Aspartate þ GTP ! fumarate þ NH3 þGDP þ Pi
Cross Several workers have demonstrated that flux through the purine
bridges nucleotide cycle increases in skeletal muscle during intense exer-
cise. This increased flux has two effects: first, it can maintain the
Ca2+
pool of adenine nucleotides, and second, it can increase the citric
Figure 1 Cycles of energy transfer in heart muscle. The central role acid cycle pool size via anaplerosis. Both of these effects are
of the Krebs cycle is highlighted. See text for details. expected to improve energy metabolism in exercising muscle.

Lactate Fatty acid

Alanine Pyruvate Acetyl-CoA Acetoacetate

Citrate
Oxaloacetate

Aspartate Isocitrate
asparagine Glutamine
Malate
Phenylalanine
tyrosine a-KETOGLUTARATE Glutamate
Valine
isoleucine
Fumarate methionine
Histidine
AMP
Proline
Succinyl-CoA Arginine
Succinate

IMP Adenylosuccinate Propionate

Odd-chain fatty acid
Aspartate GDP
GTP
Figure 2 Summary of anaplerotic pathways leading to the citric acid cycle. See text for details.
 Bioenergetics | Anaplerosis 107

Exit of Cycle Intermediates: Balancing Anaplerosis The method is illustrated in Figure 3 in which the enrichment
of the carbon skeleton of a-ketoglutarate (and therefore gluta-
Under steady-state conditions, substrates entering the citric mate, which is present in sufficient concentrations to be mea-
acid cycle via anaplerotic pathways are balanced by removing surable by NMR spectroscopy) at the C-3 and C-4 positions is
an equivalent amount of citric acid cycle intermediates through used to quantify anaplerosis. Label in these positions is gener-
pathways that maintain a constant citric acid cycle pool size. ated from randomization of the C-4 carbon in the first turn of
These pathways have been termed cataplerotic, although the the citric cycle. Therefore, the sum of the 13C enrichments of
term is somewhat an oxymoron; instead, we prefer the more the C-2 and C-3 carbons will be equal to the enrichment of the
appropriate term drainage. Drainage pathways generally involve C-4 carbon of glutamate. In contrast, with anaplerotic activity,
removing the citric acid cycle of cycle intermediates oxaloacetate the labeled C-2 and C-3 carbons will be diluted by 12C carbons
(as aspartate via transamination, or phosphoenolpyruvate via arising from unlabeled anaplerotic substrates, and the sum of
decarboxylation by phosphoenolpyruvate carboxykinase (E.C. the enrichments of the labeled C-2 and C-3 carbons will be less
4.1.1.32)), citrate via export from the mitochondrial matrix, than the enrichments of the C-4 carbon of glutamate. For
or a-ketoglutarate via transamination to glutamate. While example, using [2-13C]acetyl-CoA as a substrate and conditions
these drainage pathways are usually thought of as means to where there is no anaplerotic flux, analysis of the enrichment
balance anaplerotic pathways, they play critical roles in renal of C-2, C-3, and C-4 positions of glutamate reveals that a/c ¼ 0,
gluconeogenesis and enterocyte energy production from gluta- indicating no anaplerotic flux. Further refinements to the
mine (see below). NMR-based methods take into account the recycling of car-
bons in pyruvate (a major source of anaplerotic substrate)
derived from the citric acid cycle to improve the accuracy of
Measuring Rates of Anaplerosis measurements of anaplerotic flux.

Changes in Citric Acid Cycle Pool Size

Expression and Activity of Proteins Regulating Anaplerosis
There are two principal methods for assessing anaplerosis
based on changes in citric acid cycle pool size. The amount of The enzyme pyruvate carboxylase was discovered half a century
citric acid cycle pool intermediates can be measured enzymati- ago, and over the last decade there has been much progress in
cally or resolved with high-performance liquid chromatogra- understanding its structure and function. Using methods out-
phy. While both methods can reliably assess changes in the lined above, studies have focused on changes in enrichment of
citric acid pool size, they do not provide insight into the path- citric acid cycle intermediates to assess the activity of anaplero-
ways involved in anaplerosis nor do they determine relative tic pathways and explain changes in flux in terms of mass
rates of citric acid cycle enrichment from analperotic sub- action and allosteric regulation of key enzymes. While this is
strates. Furthermore, because anaplerosis is usually balanced most likely the case with acute changes in anaplerotic flux,
by the exit of intermediates, changes in citric acid pool size are changes in the expression and activity of enzymes responsible
generally negligible. for anaplerosis must be taken into account when studying
Simply determining changes in citric acid cycle pool size chronic processes such as pressure-overload-induced hypertro-
provides no information on rates of anaplerotic flux or on phy, heart failure, or diabetes. In streptozocin-treated diabetic
specific anaplerotic reactions. Using substrates labeled with rats, transcript levels of anaplerotic enzymes in heart and skel-
tracer 14C and measuring incorporation into specific positions etal muscle are downregulated (Figure 4). Furthermore, mice
in citric acid cycle intermediates is more revealing. This method with germline deletion of peroxiosome proliferator-activated
can determine rates of anaplerosis and is able to characterize receptor-a show an increase in pyruvate carboxylase mRNA
the pathways involved in the process. However, the method expression. Changes in transcript levels are paralleled by simi-
is also laborious and prone to error. It requires sequential lar changes in protein expression. Although, the in vitro activ-
enzymatic degradation of intermediates quantifying the release ities of the anaplerotic enzymes involved in the carboxylation
of the 14C label. This method has generally been replaced of substrates (e.g., pyruvate carboxylase) can be measured by
by 13C-NMR spectroscopy or mass spectroscopy (NMR, nuclear determining the incorporation of 14C from H14 CO1 3 , it is not
magnetic resonance). known whether changes in expression correlate with changes
in activity. However, in pressure overload-induced left ventric-
13 ular hypertrophy in the rat, compensatory increases in ana-
C-NMR/Mass Spectroscopy
plerotic flux into the citric acid cycle are accompanied by
13
C-NMR spectroscopy is used to assess quantitatively the rela- increases in transcript levels of malic enzyme.
tive contributions of various substrates to the citric acid cycle.
This method determines the anaplerotic enrichment of individ-
ual carbon positions in citric acid cycle intermediates, similar to Changes in Anaplerosis in Response to Environmental
the radiotracer method discussed before, but without the need Stress: Workload, Nutritional Status, and Disease
for digesting the carbon skeleton. 13C-labeled compounds enter
the carbon skeleton of citric acid cycle intermediates at specific Ultimately, any metabolic process has functional conse-
positions through anaplerotic pathways or through dilution of quences, and anaplerotic pathways are no exception. Anaplero-
l3
C by entry of unlabeled anaplerotic substrates. The enrichment tic pathways play important roles in regulating a wide variety
of l3C at those positions is then assessed and rates of anaplerotic of organ responses to conditions of metabolic stress ranging
flux are determined relative to rates of citric acid flux (a/c). from exercise to inborn errors of metabolism.
108 Bioenergetics | Anaplerosis

Acetyl-CoA

Citrate

Oxaloacetate

1 2 3 4 5 a-Ketoqlutarate

Without anaplerosis With anaplerosis

a-Ketoglutarate a-Ketoglutarate
and glutamate and glutamate
C2 (or C3)/C4 C2 (or C3)/C4
enrichment: 0.5 enrichment: 0.3
Figure 3 Fundamental concept of assessing anaplerotic flux using 13C-NMR spectroscopy. The black circles represent 13C that enters the citric
acid cycle pool via the first turn of the citric acid cycle. The black squares represent 13C that enters the citric acid cycle pool via the second turn of
the citric acid cycle. From Malloy C, Sherry A, and Jeffrey F (1988) Evaluation of carbon flux and substrate selection through alternate pathways
involving the citric acid cycle of the heart by 13C-NMR spectroscopy. Journal of Biological Chemistry 263: 6964–6971.

Anaplerosis in Skeletal Muscle during Exercise because the primary source of enrichment during acute exercise
appears to be flux through the reaction catalyzed by alanine
The transition from rest to moderate or intense exercise is
aminotransferase (E.C. 2.6.1.2). This reaction results in entry
associated with large increases in skeletal muscle adenosine
of glutamate as a-ketoglutarate; however, pyruvate carboxylase
triphosphate (ATP) turnover, which must be supported by up
and malic enzyme may contribute minor amounts to citric acid
to 100-fold increases in citric acid cycle flux. Although citric
cycle enrichment in exercising muscle.
acid cycle intermediates increase only three- to fourfold, these
relatively small increases in the citric acid cycle pool size (via
Glutamine Metabolism by the Small Intestine
anaplerosis) can reflect dramatic increases in citric acid cycle
flux. It has been suggested that the increases allow skeletal Glutamine is a source of energy for a number of specialized
muscle to adapt to the energetic demands of exercise, but tissues, including the small intestine. When glutamine is taken
interestingly, there is no appreciable change in citric acid up in the cell, it enters the citric acid cycle as a-ketoglutarate
cycle pool size at lower levels of exercise. The majority of and leaves the cycle through oxidation as CO2. More specifi-
changes in individual intermediates occur in the second span cally, a-ketoglutarate is converted to malate by citric acid cycle
of the citric acid cycle (i.e., from succinate to oxaloacetate) reactions and malate may be transported out of the
 Bioenergetics | Anaplerosis 109

Total protein (µg) Ischemia

5 10 20 30 40 60 80 Oxaloacetate

ACC Citrate

Succinate a-Ketoglutarate Glutamate

PC

PCC Succinyl-CoA

(a)
GDP
Soleus

Soleus
Heart

Heart
GTP
EDL

EDL
Diabetic ketoacidosis
PC Pyruvate Oxaloacetate
Citrate
(b)
Figure 4 Expression of the biotin-containing carboxylases, acetyl-CoA Succinate a-Ketoglutarate
(ACC), pyruvate carboxylase (PC), and propionyl-CoA carboxylase (PCC),
in heart muscle based on streptavidin blotting (a) and pyruvate
Succinyl-CoA
carboxylase expression in rat heart and skeletal muscle (b). Reprinted
from Gibala MJ, Young ME, and Taegtmeyer H (2000) Anaplerosis of the
citric acid cycle: Role in energy metabolism of heart and skeletal muscle.
Acta Physiologica Scandinavica 168: 657–665. GDP
GTP

Figure 5 Role of anaplerosis in improving myocardial energetics in the

mitochondria. In the cytosol, malate is converted to oxaloace- setting of ischemia (upper panel) or diabetic ketoacidosis (lower panel).
tate. The malate–aspartate shuttle is bidirectional and can also Under both conditions, loss of cofactors (NADþ/FADþ for ischemia,
transport electrons from extramitochondrial reduced nicotin- CoASH for diabetic ketoacidosis) inhibits full citric acid cycle activity.
amide dinucleotide (NADH) into the mitochondria or from Anaplerotic pathways allow the citric acid cycle to work in spans, thereby
intramitochondrial NADH to the cytosol. increasing the production of high-energy phosphates. Modified from
Taegtmeyer H and Passmore JM (1985) Defective energy metabolism of
the heart in diabetes. Lancet 1: 139–141.
Renal Ammonia Formation during Starvation
During starvation, when protein breakdown, renal gluconeo-
genesis and hepatic ketogenesis increase. Concomittantly, the operation. Recent 13C-NMR spectroscopic studies have demon-
rate of renal ammoniagenesis also increases. Ammonia is gen- strated that during myocardial ischemia, the relative contribu-
erated from amino acids, including glutamine, released by tion of anaplerosis to citric acid flux may increase sevenfold
skeletal muscle. Glutamine is converted to a-ketoglutarate in compared to nonischemic conditions. Translational research
renal cells. Alpha-ketoglutarate is metabolized to malate. based on the concept of this anaplerotic pathway has led to
Malate is transported out of the mitochondria, oxidized to the development of glutamate-enriched solutions that increase
oxaloacetate and to phosphoenolpyruvate, and ultimately the provision of anaerobic energy for the heart during coronary
used for gluconeogenesis. Although different from glutamine artery bypass surgery.
oxidation by enterocytes, this series of reactions also reflects
the balance that is generally observed between anaplerotic and Insulin Secretion, Diabetes and Ketone Body Metabolism
drainage pathways.
Insulin secretion occurs through two mechanisms. The first is a
process that involves closing of the cell-surface ATP-sensitive
Ischemia and Substrate Level Phosphorylation
potassium channels in response to increases in the circulating
Early studies assessing cardiac metabolism by measuring arte- glucose concentrations, which stimulates exocytosis of insulin-
riovenous differences in amino acid concentrations revealed containing vesicles from the b-islet cells through an increase in
that in patients with coronary artery disease and myocardial the cytosolic calcium concentration. The second mechanism is
ischemia, the heart avidly extracts glutamate and releases ala- dependent on pyruvate carboxylase, which is highly expressed in
nine. This finding led to the hypothesis that the anaplerotic b-islet cells; it has been estimated that 35–45% of pyruvate enters
substrate glutamate enters the citric acid cycle as a-ketoglutarate the citric acid cycle through this anaplerotic pathway in b-islet
via transamination with pyruvate (thereby forming the alanine cells. Inhibition of pyruvate carboxylase with phenylacetic acid
that is released). Subsequently, the a-ketoglutarate is metabo- decreases glucose-stimulated insulin release from b-islet cells. Fur-
lized to succinate with the concomitant substrate level phos- thermore, there is evidence that pyruvate carboxylase plays an
phorylation of guanosine diphosphate (GDP) to form important role in the early stages of type 2 diabetes. Specifically,
guanosine triphosphate (GTP) (Figure 5). In this way, a span in Zucker fatty rats with insulin resistance, the hyperfunctioning
of the citric acid cycle can generate high-energy phosphates in b-islet cells increase insulin production in part through
the absence of sufficient oxygen for full citric acid cycle increases in pyruvate carboxylase activity.
110 Bioenergetics | Anaplerosis

Citric acid cycle pool size increases in hearts of rats with cycle. Changes in the cell’s environment lead to depletion
experimentally induced diabetes, suggesting enrichment by and replenishment (anaplerosis) of citric acid cycle intermedi-
anaplerotic pathways. We have suggested that an increase in ates. The multiple pathways of anaplerosis utilize a variety of
anaplerotic flux, which primarily occurs through pyruvate car- substrates, including carbohydrates, odd-chain fatty acids, and
boxylation (via malic enzyme), plays an important role in amino acids. These pathways reflect a system of redundancy
maintaining flux through the second span of the citric acid that is important for both function and survival of the cell.
cycle. Acutely, the metabolic derangement of ketoacidosis
that occurs with diabetes inhibits flux through a-ketoglutarate
dehydrogenase by sequestration of coenzyme A (CoASH). This See also: Bioenergetics: Mitochondrial Metabolite Carrier Protein
phenomenon is associated with contractile dysfunction of the Family; Metabolism Vitamins and Hormones: Amino Acid
heart that can be readily reversed by the addition of glucose, Metabolism; Branched-Chain Amino Acids.
lactate, or pyruvate (all of which are anaplerotic substrates).
The effects of pyruvate are mediated by enrichment of malate
in the citric acid cycle pool, which occurs by carboxylation of Further Reading
pyruvate to form malate and oxaloacetate through the actions of
malic enzyme and pyruvate carboxylase, respectively (Figure 5). Baldwin JE and Krebs HA (1981) The evolution of metabolic cycles. Nature
The citric acid cycle is thereby able to operate once again in a 291: 381–382.
Cohen DM and Bergman RN (1997) Improved estimation of anaplerosis in heart using
span that can generate reducing equivalents to support oxidative 13
C NMR. American Journal of Physiology 273: E1228–E1242.
phosphorylation of adenosine diphosphate to form the ATP Gibala MJ, Young ME, and Taegtmeyer H (2000) Anaplerosis of the citric acid cycle:
necessary to drive the contractile machinery of the heart. Role in energy metabolism of heart and skeletal muscle. Acta Physiologica
Scandinavica 168: 657–665.
Gunawardana SC, Liu YJ, Macdonald MJ, Straub SG, and Sharp GW (2004) Anaplerotic
Long-Chain Fatty Acid Oxidation Defects and Myopathies input is sufficient to induce time-dependent potentiation of insulin release in rat
pancreatic islets. American Journal of Physiology – Endocrinology and Metabolism
The inability to oxidize long-chain fatty acids due to deficien- 287: E828–E833.
cies in activity of carnitine palmitoyltransferase-1 (E.C. Kornberg H (1966) Anaplerotic sequences and their role in metabolism. Essays in
2.3.1.21) or the enzymes involved in b-oxidation is associated Biochemistry 2: 1–31.
Krebs HA and Johnson WA (1937) The role of citric acid in intermediate metabolism in
with contractile dysfunction due to skeletal and heart muscle animal tissues. Enzymologia 4: 148–156.
damage. One proposed mechanism responsible for the Malloy C, Sherry A, and Jeffrey F (1988) Evaluation of carbon flux and substrate
decrease in contractile activity is decreased citric acid cycle selection through alternate pathways involving the citric acid cycle of the heart by
13
flux due to loss of intermediates from damaged myocytes. C NMR spectroscopy. Journal of Biological Chemistry 263: 6964–6971.
Martini WZ, Stanley WC, Huang H, et al. (2003) Quantitative assessment of anaplerosis
Based on this hypothesis, one clinical study has treated patients
from propionate in pig heart in vivo. American Journal of Physiology
with defects in long-chain fatty acid oxidation with odd-chain 284: E351–E356.
triglycerides (which can increase the citric acid cycle pool size Mochel F, DeLonlay P, Touati G, et al. (2005) Pyruvate carboxylase deficiency: Clinical
by entering as succinyl-CoA) and tested the hypothesis that an and biochemical response to anaplerotic diet therapy. Molecular Genetics and
increase in citric acid cycle may improve muscle function. As Metabolism 84: 305–312.
Owen OE, Kalhan SC, and Hanson RW (2002) The key role of anaplerosis and
proof of principle, the study has demonstrated beneficial cataplerosis for citric acid cycle function. Journal of Biological Chemistry 277(34):
effects such as reversing left ventricular dysfunction, decreasing 30409–30412.
muscle breakdown, and decreasing weakness. However, the Panchal AR, Comte B, Huang H, et al. (2000) Partitioning of pyruvate between oxidation
effects of medium-length odd-chain fatty acids as potential and anaplerosis in swine hearts. American Journal of Physiology 279(5):
H2390–H2398.
anaplerotic substrates for the treatment of other forms of
Pound KM, Sorokina N, Ballal K, et al. (2009) Substrate–enzyme competition attenuates
heart failure have not yet been assessed. upregulated anaplerotic flux through malic enzyme in hypertrophied rat heart and
Pyruvate carboxylase deficiency is a rare autosomal reces- restores triacylglyceride content: Attenuating upregulated anaplerosis in
sive disease that affects a variety of organs, including the liver, hypertrophy. Circulation Research 104: 805–812.
kidney, brain, skeletal muscle, heart, and adipose tissue. The Roe CR, Sweetman L, Roe DS, David F, and Brunengraber H (2002) Treatment of
cardiomyopathy and rhabdomyolysis in long-chain fat oxidation disorders using an
disorder is characterized by impaired gluconeogensis, lactic anaplerotic odd-chain triglyceride. Journal of Clinical Investigation 110: 259–269.
acidosis, and, depending on the severity of the disease, neuro- Russell R and Taegtmeyer H (1991) Changes in citric acid cycle flux and anaplerosis
logic manifestations ranging from minimal neurologic antedate the functional decline in isolated rat hearts utilizing acetoacetate. Journal of
abnormalities to profound psychomotor retardation. In the Clinical Investigation 87: 384–390.
Russell R and Taegtmeyer H (1991) Pyruvate carboxylation prevents the decline in
most severe form of the disease, patients die within the first
contractile function of rat hearts oxidizing acetoacetate. American Journal of
few weeks of life. Nutritional treatment of the disease with Physiology 261: H1756–H1762.
anaplerotic odd-chain triglycerides, such as triheptanoin, or Sorokina N, O’Donnell JM, McKinney RD, et al. (2007) Recruitment of compensatory
aspartate has been shown to prolong life. pathways to sustain oxidative flux with reduced carnitine palmitoyltransferase I
activity characterizes inefficiency in energy metabolism in hypertrophied hearts.
Circulation 115: 2033–2041.
Summary and Perspective
Efficient energy transfer in the mammalian cell is linked to a
series of moiety-conserved cycles, including the citric acid
 Arachidonic Acid Regulated Calcium Channel
T J Shuttleworth, University of Rochester Medical Center, Rochester, NY, USA
ã 2013 Elsevier Inc. All rights reserved.

Glossary STIM1 (stromal interacting molecule 1) A protein that was

AKAP (A-kinase anchoring protein) A family of proteins originally identified as a cell-surface protein that played a
that binds to the regulatory subunits of the cyclic key role in interactions between hematopoietic cells and
AMP-activated kinase protein kinase A, to provide a scaffold cells in the bone marrow stroma, and as a growth suppressor
enabling the targeting of various other proteins of certain tumors. More recently, it was identified as
(e.g., kinases, phosphodiesterases, phosphatases, etc.) playing an essential role in the activation of store-operated
in close proximity to PKA, whose activity they regulate. Ca2þ entry via its ability to sense falling Ca2þ concentrations
ARC channel (arachidonic acid regulated Ca2þ within the intracellular stores and to subsequently activate
channel) A small conductance, highly Ca2þ-selective the plasma-membrane store-operated Ca2þ channels.
channel that is specifically activated by low concentrations Subsequent studies have revealed that the pool of STIM1
(2–8 mM) of arachidonic acid in a manner that is entirely that constitutively resides in the plasma membrane plays
independent of any store deletion (cf. CRAC channels). an equally critical role in the activation of the
CRAC channel (Ca2þ release-activated Ca2þ store-independent ARC channels.
channel) Another small conductance, highly Ca2þ-selective TRPC channels (canonical TRP channels) A subgroup of
channel that is specifically activated following depletion the mammalian transient receptor potential (TRP) channels
of intracellular Ca2þ stores. that is most closely related to the TRP channels of Drosophila.
Orai A group of three recently identified novel proteins They are essentially nonselective cation channels that are
(Orai1–3) that are the pore-forming components of both activated under various conditions of receptor activation,
CRAC and ARC channels. some of which are claimed to be store-operated and to
involve STIM1, although this remains controversial.

Different Modes of Ca2+ Entry and the Discovery raises several questions as to its ability to function as the
of the ARC Channels exclusive pathway of agonist-activated Ca2þ entry in other
cell types. These include a requirement for a rather profound
The receptor-activated entry of Ca2þ in nonexcitable cells plays a depletion of intracellular stores before any activation begins,
key role in the generation and modulation of the resulting the subsequent relatively slow rate of activation, and evidence
intracellular Ca2þ signals that are known to be responsible for indicating that such activation occurs in an essentially all-or-
a wide variety of critical cellular responses, including cell growth none fashion. Together, these features are difficult to reconcile
and proliferation, differentiation, movement, secretion, metab- with the predicted requirement of a finely regulated, graded
olism, and cell death. Our understanding of the underlying Ca2þ response in order to accurately coordinate the appropriate
mechanisms and pathways involved in such entry began with cellular response to any particular increase in the Ca2þ signal.
the work of Putney in the mid-1980s and his development On a more general level, it should also be remembered that
of the concept of capacitative, or store-operated, Ca2þ entry, depletion of the intracellular Ca2þ stores can be induced by a
where activation of the entry of Ca2þ is strictly a consequence variety of different events, many of which are unrelated to the
of the depletion of intracellular Ca2þ stores. Demonstration of normal, physiologically relevant, agonist-activated signaling
the presence of such store-operated entry pathways in almost all pathways. For example, store depletion can result from an inhi-
cell types examined has led to the widely held belief that this bition of the sarcoplasmic and endoplasmic calcium (SERCA)
specific mode of Ca2þ entry represents the major, if not the only, pump such as may occur during ischemia, or an increase in
mode of such receptor-activated entry in nonexcitable cells. passive leak pathways such as those provided by the presenilins,
However, examination of the literature reveals that direct evi- as well as exposure to oxidants and other cellular stressors.
dence demonstrating a principal role for such pathways in the Finally, the absolute requirement for a sustained depletion of
various receptor-activated Ca2þ signals and responses seen in intracellular Ca2þ stores in order to activate these conductances
these different cell types is limited. More specifically, although is potentially problematic, as such depletion can seriously
various conductances have been proposed to provide the route impact various vital cell processes, the most obvious being the
for store-operated Ca2þ entry in different cells types, the most correct folding and processing of secretory and membrane pro-
thoroughly characterized store-operated Ca2þ channel remains teins. Together, consideration of these issues suggests that,
the Ca2þ-release activated Ca2þ (CRAC) channel. This conduc- although store-operated modes of Ca2þ entry can be demon-
tance was originally identified in T lymphocytes and in mast strated in almost all cell types, their principal role might lie in
cells, and here it is clear that the activity of the CRAC channels is some process other than normal signaling in response to agonist
critical for the physiological activation of these cells. However, stimulation. Indeed, given their exclusive dependence on the
examination of the properties of this channel and its activation depletion of intracellular Ca2þ stores for activation, the most

127
128 Bioenergetics | Arachidonic Acid Regulated Calcium Channel

likely primary role for such store-operated entry pathways would 0.1
seem to be the maintenance of adequate levels of Ca2þ in the ER. Time (s)
0

Inward current at −80 mV (pA/pF)

These arguments eventually led to the search for possible
0 50 100 150 200
alternate pathways of agonist-activated Ca2þ entry whose activ-
−0.1
ity might be more directly or specifically connected to agonist-
induced receptor activation. The result of one such search was −0.2
the discovery of an entry pathway whose activation is entirely
unaffected by store depletion and is instead dependent on the −0.3
agonist-activated, receptor-mediated generation of arachidonic
acid. Initial evidence in support of such a pathway included the −0.4
demonstration that addition of low concentrations (<8 mM)
of exogenous arachidonic acid resulted in an entry of Ca2þ −0.5

without detectible release from stores, that the generation of

−0.6
arachidonic acid was increased following addition of low phys- (a)
iologically relevant concentrations of agonists to cells, and that
mV
the pharmacological inhibition of such agonist-activated ara- 0
chidonic acid generation resulted in the inhibition of the asso- −100 −60 −20 20 60
ciated entry of Ca2þ. Finally, it was shown that the activation of −0.2
the entry of Ca2þ was an effect of arachidonic acid itself, and not
any of its metabolites. Subsequently, much as the CRAC chan- −0.4
pA/pF
nel was eventually identified as the archetypal store-operated
Ca2þ-selective conductance, the specific conductance responsi- −0.6
ble for this store-independent, arachidonic acid-activated Ca2þ
entry was identified and named the arachidonic acid regulated (b) −0.8
Ca2þ (ARC) channel. In the approximately 10 years since their
first identification, considerable details of these channels have
been revealed, including their biophysical and pharmacological
profiles, specificity for arachidonic acid, occurrence in different
cell types, and, most recently, the molecular basis of their com-
position and regulation. Moreover, there are now clear exam-
(c)
ples of situations where these ARC channels have been shown
to play unique, critical roles in the physiological responses of Figure 1 Basic features of currents through endogenous ARC
various cell types. channels. (a) Whole-cell patch-clamp recording of inward current density
at 80 mV in a HEK293 cell. Free Ca2þ concentration in the external
(bath) solution was 10 mM Ca2þ and in the internal (pipette) solution was
100 nM. At the point indicated (arrow), 8 mM arachidonic acid was
Characteristics of ARC Channels
added to the external bath. (b) Representative current–voltage
relationship of the arachidonic acid-activated current showing the
Much like the CRAC channels, ARC channels represent a rela- marked inward rectification and very positive reversal potential typical of
tively small (with typical whole-cell currents of approximately a highly Ca2þ-selective conductance. (c) Comparison of the current
0.5–1.5 pA pF1 in most cell types), highly Ca2þ-selective con- traces through the ARC channels (left) and through the corresponding
ductances. Consistent with this high selectivity for Ca2þ, their store-operated CRAC channels (right) present in the same cells
current–voltage relationship reveals marked inward rectifica- during brief (250 ms) pulses to 120 mV. Note that the fast-inactivation
tion and a reversal potential in excess of þ60 mV (Figure 1). characteristic of currents through CRAC channels is absent in the
Similarly, like most Ca2þ channels, they are blocked by La3þ ARC channel currents.
and by Gd3þ with complete inhibition achieved by 50–100 mM
and 5 mM, respectively. However, unlike the CRAC channels, as it is known that arachidonic acid at higher concentrations
they are not affected by moderate acidification of the external (e.g., >10–20 mM) can induce significant effects on the integ-
medium, and they are unaffected by the widely used, but rity of the cell membrane, resulting in the permeation of even
relatively nonspecific, drug 2-aminoethoxydiphenyl borate relatively large, normally impermeant, ions such as NMDGþ.
(2-APB). In addition, the ARC channels do not share the clear Activation of the ARC channels also shows a high specificity
fast inactivation during brief pulses to negative potentials that for arachidonic acid, as both saturated fatty acids (e.g., palmitic
are a characteristic of CRAC channels (Figure 1). acid) and mono-unsaturated fatty acids (e.g., oleic acid) are
Activation of the ARC channels occurs at the same low completely without effect. Even among other polyunsaturated
concentrations of exogenous arachidonic acid (2–8 mM) as fatty acids, only linoleic acid shows a small effect and this proba-
was seen to induce store-independent Ca2þ entry in the earlier bly arises from the fact that this fatty acid is a metabolic precursor
studies (see above). Importantly, such concentrations are for arachidonic acid. In addition, and in contrast to certain
clearly physiologically relevant as they are similar to values members of the transient receptor potential channel (TRPC)
for the Kd’s of the cytosolic enzymes that metabolize arachi- family of channels, the ARC channels are entirely unaffected by
donic acid. Moreover, effective and specific activation of the diacylglycerol and its analogs. Finally, experiments using the
conductance by these low concentrations is a critical property nonpermeable arachidonic acid analog arachidonyl-coenzyme
 Bioenergetics | Arachidonic Acid Regulated Calcium Channel 129

A demonstrated that the activation of the channel by arachidonic studies utilizing a cell-permeable stearated version of a synthetic
acid is exclusively via an action of the fatty acid at the inner face of peptide that disrupts the binding of PKA to AKAPs (st-Ht31
the membrane. peptide) both inhibited normal ARC channel currents and
Since their initial discovery and characterization almost blocked the above effects of forskolin on the currents.
10 years ago, ARC channels (or, at least, conductances display-
ing all the key features of these channels) have been described in
a wide variety of different cell types, including many common Molecular Nature (and Activation) of ARC Channels
cell lines (HEK293, HeLa, COS, and RBL), SY6Y neuroblastoma
cells, K562 erythroleukemia cells, as well the exocrine parotid Although the CRAC channel had been originally described and
and pancreatic acinar cells, and the endocrine pancreatic b cells. characterized in the early 1990s, the molecular nature of its
In many of these cell types, the ARC channels coexist with store- structure and regulation were unknown until the recent identifi-
operated channels, including the biophysically very similar cation of stromal interacting molecule 1 (STIM1) as the sensor
CRAC channels. Nevertheless, it can be demonstrated that of store depletion, and the discovery of Orai1 as the molecule
these two coexisting channels represent entirely distinct entities. that forms the CRAC channel itself. These studies revealed that
Thus, using the whole-cell patch clamp mode in HEK293 cells, it STIM1 located in the membrane of the endoplasmic reticulum
has been shown that maximal activation of the CRAC channels (ER) sensed the depletion of Ca2þ in these stores via a luminal
by store depletion has no effect on the magnitude of the subse- Ca2þ-binding EF-hand motif present in the N-terminal region of
quently activated ARC channel currents induced by addition of the protein. Loss of Ca2þ from this motif results in the oligo-
exogenous arachidonic acid – in other words, at least under merization of STIM1 and its translocation to regions of the ER
whole-cell patch clamp conditions, the two currents are strictly that lie adjacent to the plasma membrane. Here, it interacts with
additive in the same individual cell. Orai1 resulting in the activation of the CRAC channels.
Given this clearly demonstrated role of STIM1 as the ER
Ca2þ sensor in the activation of the CRAC channels, it would
Reciprocal Regulation of ARC Channels and be expected that STIM1 would be unlikely to play any role in
CRAC Channels the activation of the store-independent ARC channels. How-
ever, experiments showed that ARC channel currents were
As described above, under whole-cell patch clamp conditions, increased by overexpression of STIM1 and reduced by siRNA
currents through the ARC channels and coexisting store- knock-down of the endogenous protein, in a manner that was
operated CRAC channels are strictly additive. However, it is indistinguishable from that seen with the CRAC channels pres-
important to note that such additivity of the two currents does ent in the same cells. Reconciliation of these unexpected results
not appear to occur in the intact cell. The reason for this began with the realization that the STIM1 protein is not exclu-
difference is that, in the whole-cell patch clamp configuration, sively restricted to the ER membrane. In fact, it was originally
the presence of buffers in the pipette solution clamps the intra- identified as a cell surface (i.e., plasma-membrane) protein
cellular Ca2þ concentration at resting levels while, in the normal present in the stromal cells of the bone marrow, where it was
intact cell situation, activation of store-operated Ca2þ entry typi- involved in interactions between these cells and hematopoietic
cally results in a sustained elevation of intracellular Ca2þ. Studies cells (specifically pre-B lymphoidal cells). Studies have shown
have shown that this sustained elevation in intracellular Ca2þ that the pool of STIM1 that is resident in the plasma membrane
results in the activation of the Ca2þ-calmodulin-dependent is relatively small (approximately 10–25% depending on cell
phosphatase calcineurin and this, in turn, induces an inhibition type), and its delivery to this location is strictly dependent
of ARC channel activity. Consequently, in the intact cell, activa- on N-linked glycosylation at two sites within the N-terminal
tion of the store-operated channels (e.g., CRAC channels) will region of the protein (Asn131 and Asn171). To examine the
result in an inhibition of the ARC channels, a process that has effect of specifically eliminating this cell-surface pool of STIM1,
been described as the ‘reciprocal regulation of Ca2þ entry’. cells treated with an siRNA targeting the endogenous STIM1
The discovery that the phosphatase calcineurin inhibits the were transfected with an siRNA-resistant STIM1 construct in
ARC channels suggested that the activity of these channels must which these two glycosylation sites were mutated (N131Q and
be dependent, in some way, on phosphorylation. Subsequent N171Q) – thereby effectively replacing the native STIM1 with
studies showed that the specific kinase responsible was the cyclic the mutant version. Examination of both ARC and CRAC
AMP-dependent protein kinase A (PKA). Thus, the currents currents in these cells revealed that while CRAC channel cur-
through the ARC channels measured in HEK293 cells main- rents were unaffected, the ARC channel currents in the same
tained under normal culture conditions following addition of cells were profoundly inhibited (Figure 2). These data clearly
low concentrations of arachidonic acid are inhibited by pre- established that it is the pool of STIM1 resident in the plasma
incubation with the cell-permeable PKA-specific inhibitory pep- membrane that is essential for activation of the ARC channels.
tide myristolated PKI but are unaffected by inhibition of either As yet, exactly how the plasma membrane STIM1 acts to regu-
protein kinase C (with calphostin) or protein kinase G (with late the activation of the ARC channels is unknown. However,
KT5823). Correspondingly, the addition of forskolin, particu- it is important to note that, in the case of this plasma mem-
larly after induction of the calcineurin-mediated dephosphory- brane pool of STIM1, the Ca2þ-binding N-terminal EF-hand
lation, resulted in enhanced arachidonic acid-activated ARC motif will lie in the extracellular environment. Given the high
channel currents. In addition, it has been shown that this action and relatively stable Ca2þ concentrations of this environment,
of PKA in modulating ARC channel activity involves a member it is clear that the EF-hand of this pool of STIM1 would always,
of the A-kinase anchoring protein (AKAP) family. For example, under normal circumstances, be in the Ca2þ-bound state.
 130 Bioenergetics | Arachidonic Acid Regulated Calcium Channel

cont K/D K/D + mut Orai3 are required for the formation of the functional ARC
channel. Thus, overexpression of Orai1 in cells stably expressing
100
STIM1 results, not only in markedly increased CRAC channel
STIM1
currents but also in similar increases in ARC channel currents.
Correspondingly, expression of the dominant-negative (E106Q)
75 Orai1 mutant effectively eliminates both CRAC and ARC
Deglycos-STIM1
channel currents. However, expression of the corresponding
dominant-negative E81Q Orai3 mutant essentially eliminates
(a)
currents through the ARC channels and has no effect on the
0.8 0.8 CRAC channel currents in the same cells.

0.7 0.7
Inward current @ −80 mV (pA/pF)

Inward current @ −80 mV (pA/pF)

ARC Channel Structure
0.6 0.6
Recent studies, utilizing a variety of different approaches, have
0.5 0.5
demonstrated that the functional CRAC channel is a tetrameric
0.4 0.4 assembly of Orai1 subunits. Given this, and the above data
demonstrating the essential requirement for both Orai1 and
0.3 0.3 Orai3 subunits in the functional ARC channel, the most likely
structure for the channel would seem to be some form of
0.2 0.2 heterotetrameric assembly of Orai1 and Orai3 subunits. How-
ever, examination of the conductances resulting from each of
0.1 0.1
the possible tetrameric combinations of Orai1 and Orai3 sub-
0 0 units expressed in the form of pre-assembled concatenated
(b) W/T Mutant W/T Mutant constructs revealed unexpected results. Thus, while expression
of some of these constructs resulted in significant arachidonic
Figure 2 Expression of STIM1 in which the glycosylation sites that are acid-activated, highly Ca2þ-selective currents, they also gave
essential for delivery of the protein to the plasma membrane are rise to similarly increased highly Ca2þ-selective currents that
mutated reveals that activation of ARC channels is exclusively dependent
were activated by store depletion. Such a mixed selectivity in
on the pool of STIM1 that constitutively resides in the plasma membrane.
(a) Western blot of STIM1 in HEK293 cells, showing the levels of total
their mode of activation is entirely inconsistent with the native
STIM1 in untransfected cells (cont), in cells transfected with an STIM1 channels and, instead, suggests that such constructs generate
siRNA construct (K/D), and in siRNA-treated cells following transfection some form of hybrid CRAC/ARC channel. Subsequently, addi-
with an siRNA-resistant STIM1 in which the two N-glycosylation sites have tional experiments revealed that the functional ARC channel is,
been mutated (N131Q and N171Q) (K/D þ mut). (b) The effects of in fact, a pentameric structure formed from three Orai1 subunits
expressing siRNA-resistant versions of either wild-type STIM1 (gray bars) and two Orai3 subunits (Figure 3). Expression of pre-assembled
or the glycosylation-mutant STIM1 (black bars) in STIM1 siRNA-treated concatenated pentameric constructs with this composition
cells. Currents through the store-operated CRAC currents (left panel) are resulted in large arachidonic acid-activated Ca2þ-selective cur-
unaffected by the expression of the glycosylation-mutant STIM1, while rents without affecting the corresponding store-operated currents
similar expression of the glycosylation-mutant STIM1 results in a profound
(Figure 3). Critically, the arachidonic acid-activated currents
inhibition of ARC channel currents (right panel). Reproduced from Mignen
O, Thompson JL, and Shuttleworth TJ (2007) STIM1 regulates Ca2þ entry
resulting in the expression of these pentameric constructs dis-
via arachidonate-regulated Ca2þ-selective (ARC) channels without store- played all the key biophysical and pharmacological features of
depletion or translocation to the plasma membrane. Journal of Physiology the endogenous ARC channels, including a high sensitivity to low
579: 703–715. concentrations (2–4 mM) of exogenous arachidonic acid, insen-
sitivity to 2APB, and even an exclusive dependence on the
plasma-membrane pool of STIM1 for their activation (Figure 4).
This stands in marked contrast to the critical role of the loss of As noted above, this demonstration that the functional ARC
Ca2þ from the luminal EF-hand of STIM1 in the ER in the channel was a pentameric structure was unexpected, given that
regulation of the store-operated CRAC channels. Conse- it had previously been shown that the functional CRAC channel
quently, whatever the mechanism by which STIM1 regulates was a tetramer. It remains to be determined precisely what role, if
the ARC channels, it must be very different from the way it any, this fundamental difference in the overall structure of the
activates the CRAC channels. two channels plays in the determination of their individual spe-
The demonstration that STIM1 is required for the activation cific properties and distinct modes of activation.
of both CRAC and ARC channels, although entirely distinct
pools of this protein are responsible, and that both channels
display very similar biophysical properties raised the possibil- What Do ARC Channels Do?
ity that, as had already been demonstrated for the CRAC chan-
nels, Orai proteins may form the ARC channel. Three distinct, The fact that the ARC channels coexist with store-operated
but closely related Orai proteins (Orai1–3) are expressed in conductances in the same cells, and their biophysical proper-
mammals, and while the CRAC channel is formed exclusively ties are generally so similar to the store-operated CRAC chan-
from Orai1, studies have demonstrated that both Orai1 and nels, raises the obvious question of what their specific role
 Bioenergetics | Arachidonic Acid Regulated Calcium Channel 131

under these conditions, it is the ARC channels that provide

3 3 the predominant pathway for Ca2þ entry. Although occasional
1 3 1 1 arguments to the contrary have been proposed, these have
generally relied on pharmacological approaches which are
severely limited by the questionable specificity and selectivity
1 1 1 3
of the drugs currently available (e.g., 2-APB), or have involved
(a) the application of arachidonic acid at concentrations that are
known to induce obvious nonspecific effects on membrane
−3.5 integrity. Clearly, definitive identification of the specific Ca2þ
entry pathways operating during such oscillatory responses can
−3 only come from studies involving the direct electrophysiologi-
Current at −40 mV (pA/pF)

cal analysis of the activated conductance(s). Using this crite-

−2.5
rion, studies in HEK293 cells, parotid acinar cells, and in
pancreatic acinar cells have all confirmed that the only Ca2þ
−2
entry channel that can be demonstrated to be active under such
conditions is the store-independent ARC channel. Such analy-
−1.5
sis is particularly clear in the parotid and pancreatic acinar cells,
−1 as the biophysical properties of the store-operated conduc-
tances in these cells are entirely distinct from those of the ARC
−0.5 channels – they are relatively nonselective conductances (most
likely reflecting the activity of TRPC channels), displaying rela-
0 tively linear current–voltage relationships and with obvious
(b) Control 31113 31311 outward currents at positive potentials. Moreover, in the case
of the pancreatic acinar cells, the precise physiologically rele-
mV mV vant concentrations of cholecystokinin, a major endogenous
0 0
−100 −50 0 50 100 −100 −50 0 50 100
activator of secretion, are known (10–20 pM), and studies have
−2 −2
demonstrated that the only detectible Ca2þ entry conductance
activated at these concentrations is the ARC channel.
−4
pA/pF −4 pA/pF A recent report has identified a more specific, and poten-
−6 tially highly important, role of the ARC channels as the key
−6 player in the release of insulin from pancreatic b cells. Thus,
−8
insulin secretagogs such as acetylcholine and cholecystokinin
(c) −10 −8
are known to induce the generation of arachidonic acid which,
Figure 3 The functional ARC channel pore is comprised of a in turn, triggers insulin secretion from islets at low glucose
heteropentamer of three Orai subunits and two Orai3 subunits. levels and enhances the secretion of insulin mediated by depo-
(a) Cartoon illustrating the structural organization of the two Orai larization. The recent study revealed that this action of arachi-
heteropentamers that have been shown to duplicate all the features of the donic acid did not involve voltage-gated Ca2þ channels and
endogenous ARC channels. (b) Inward currents, measured at 40 mV, instead specifically resulted from the activation of ARC chan-
activated by addition of 8 mM exogenous arachidonic acid in nels in these cells. The ARC channels therefore represent a
untransfected cells stably expressing STIM1 (control) are increased critical, and previously unsuspected, component of the overall
8–10-fold following expression of either the 31113 or 31311 pentamer
secretagog-induced release of insulin in pancreatic b cells.
in the form of pre-assembled concatenated constructs. (c) Representative
current–voltage relationships of the arachidonic acid-activated currents
in STIM1-stable cells expressing either the concatenated 31113 pentamer
(left) or the 31311 pentamer (right). Both display the marked inward Conclusions and Implications
rectification and very positive reversal potential seen in the endogenous
ARC channels. The demonstrated existence of the ARC channels in a range
of different cells types, and the store-independent agonist-
activated Ca2þ entry pathway that they provide, has several
might be. The most likely answer was that their role is related, important implications. The most obvious and important is
in some way, to their unique store-independent mode of acti- that it can no longer be assumed that simply demonstrating
vation and this has turned out to be the case. Thus, it has been that store-operated Ca2þ entry is present in a cell (e.g., by
demonstrated in various cell types that the ARC channels are addition of thapsigargin) implies that it is the only pathway
specifically activated by the low, physiologically relevant, con- for the agonist-activated entry of Ca2þ entry in that cell. Deter-
centrations of agonists that typically produce Ca2þ signals in mination of which specific Ca2þ entry pathway is operating
the form of repeated oscillations of various frequencies. Studies under the relevant conditions can only reliably be achieved by
have shown that such oscillatory Ca2þ signals are associated the direct measurement, and biophysical analysis, of the con-
with, at most, only a minimal (2–5%) depletion of the intra- ductance(s) active under those conditions. The necessity of
cellular Ca2þ stores – levels that would not be capable of such specific identification of the relevant Ca2þ entry pathway
inducing activation of the store-operated conductances (see is only emphasized by the fact that, as discussed above, the
above). Consequently, the available evidence indicates that, ARC channels have been shown to be present in a wide variety
132 Bioenergetics | Arachidonic Acid Regulated Calcium Channel

−3

mV
0
−100 −60 −20 20 60
−2
−2

pA/pF
2 µM

−1 −4 pA/pF

4 µM
−6
8 µM
0
0 2 4 6 8 10 −8
(a) AA conc. (µM) (b)

−1.75
mV
0
−100 −60 −20 20 60 −1.5

Gd3+ −2

Current at −40 mV (pA/pF)

+
−1.25

−4 pA/pF
−1

−6 −0.75

−8 −0.5
(c)
−0.25

0
(d) 31311 31113

Figure 4 The features of the currents induced by expression of the appropriate Ora1/Orai3 heteropentamers precisely duplicate those of the
endogenous ARC channels. (a) Activation of the currents induced following expression of the concatenated 31311 pentamer in STIM1-stable cells
occurs at the same low concentrations of exogenous arachidonic acid as that reported for the endogenous ARC channels. Shown are the mean  SE
of the inward currents at 40 mV activated by the indicated concentrations arachidonic acid. (b) The resulting current–voltage relationships at all
concentrations of arachidonic acid display marked inward rectification and very positive (>þ 60 mV) reversal potentials. (c) The arachidonic
acid-activated currents in STIM1-stable cells expressing the concatenated 31311 heteropentamer are completely blocked by 5 mM gadolinium (Gd3þ).
(d) Activation of the increased arachidonic acid-induced currents seen on expression of the 31113 and 31311 pentamers is entirely dependent on STIM1
in the plasma membrane. Shown are inward current density at 40 mV in STIM1-stable cells treated with siRNA to STIM1, and transfected with a
wild-type STIM1 (white bar), together with the same siRNA-treated cells expressing either the 31311 or 31113 concatenated pentamer, as indicated,
after transfection with either an siRNA-resistant wild-type STIM1 (black bars) or with an siRNA-resistant glycosylation mutant STIM1 (gray bars).
Reproduced from Mignen O, Thompson JL, and Shuttleworth TJ (2009) The molecular architecture of the arachidonate-regulated Ca2þ-selective
ARC channel is a pentameric assembly of Orai1 and Orai3 subunits. Journal of Physiology 587: 4181–4197.

of different cell types, and their potential to provide a unique, manner would likely be profoundly impacted by the relative
store-independent, route for agonist-activated Ca2þ entry in levels of expression of either of these components – resulting
such cells is clear. channels of unknown composition and/or stoichiometry. In
An additional point to note is that the simple demonstra- addition to Orai1 and Orai3, the ARC channels also require
tion of a dependence on STIM1 and/or Orai1 for any particular adequate STIM1 in the plasma membrane for their activation.
cellular response or effect is not sufficient to definitively con- Limitations in any one of these components would be expected
clude that such a response involves store-operated Ca2þ entry, to have significant impact on overall channel activity. Moreover,
or the activity of CRAC channels, as both STIM1 and Orai1 as discussed above, studies have shown that ARC channel activ-
proteins are also essential for the activity of the ARC channels. ity is also dependent on a balance between a PKA-dependent
Consequently, proposals to target these proteins therapeuti- phosphorylation which requires an AKAP and a calcineurin-
cally as a means of specifically affecting CRAC channel activity mediated dephosphorylation. Consequently, factors that influ-
are not valid. In addition, it is clear that caution must be ence the balance between these opposing actions would likely
applied to studies involving the simple overexpression of indi- profoundly impact the resulting entry of Ca2þ through the
vidual components of the overall ARC channel complex and its channels, with the obvious effects on the resulting agonist-
regulators. Thus, we already know that the ARC channel induced Ca2þ signals generated in the cell.
requires both Orai1 and Orai3 subunits for its formation, but As discussed above, the specific properties and features
the ability of these subunits to assemble in the appropriate of the ARC channels suggest that they are uniquely suited to
 Bioenergetics | Arachidonic Acid Regulated Calcium Channel 133

provide a critical role in agonist-activated Ca2þ entry, particularly Mignen O, Thompson JL, and Shuttleworth TJ (2005) Arachidonate-regulated
under conditions where the sustained depletion of intracellular Ca2þ-selective (ARC) channel activity is modulated by phosphorylation and
involves an A-kinase anchoring protein. Journal of Physiology 567: 787–798.
Ca2þ stores is either unlikely or inappropriate. Consistent with
Mignen O, Thompson JL, and Shuttleworth TJ (2007) STIM1 regulates Ca2þ entry
this, the activity of these channels has been shown to play a key via arachidonate-regulated Ca2þ-selective (ARC) channels without store-
role in important physiological responses of diverse cell types, depletion or translocation to the plasma membrane. Journal of Physiology 579:
including the insulin-secreting b cells, and the exocrine cells of 703–715.
the parotid and pancreas. Undoubtedly, as more groups begin to Mignen O, Thompson JL, and Shuttleworth TJ (2008) Both Orai1 and Orai3 are
essential components of the arachidonate-regulated Ca2þ-selective ARC channels.
study these channels, additional roles will be revealed. Journal of Physiology 586: 185–195.
Mignen O, Thompson JL, and Shuttleworth TJ (2009) The molecular architecture
of the arachidonate-regulated Ca2þ-selective ARC channel is a pentameric
See also: Bioenergetics: ORAI Store-Operated Calcium Channel; assembly of Orai1 and Orai3 subunits. Journal of Physiology 587: 4181–4197.
Store-Operated Calcium Channels. Shuttleworth TJ (1999) What drives calcium entry during [Ca2þ]i oscillations?
Challenging the capacitative model. Cell Calcium 25: 237–246.
Shuttleworth TJ (2009) Arachidonic acid, ARC channels, and Orai proteins.
Cell Calcium 45: 602–610.
Further Reading Shuttleworth TJ, Thompson JL, and Mignen O (2004) ARC channels – a novel pathway
for receptor-activated calcium entry. Physiology 19: 355–361.
Dziadek MA and Johnstone LS (2007) Biochemical properties and cellular localisation Shuttleworth TJ, Thompson JL, and Mignen O (2007) STIM1 and the noncapacitative
of STIM proteins. Cell Calcium 42: 123–132. ARC channels. Cell Calcium 42: 183–191.
Manji SSM, Parker NJ, Williams RT, et al. (2000) STIM1: A novel phosphoprotein Wolf BA, Turk J, Sherman WR, and McDaniel ML (1986) Intracellular
located at the cell surface. Biochimica et Biophysica Acta 1481: 147–155. Ca2þmobilization by arachidonic acid. Comparison with myo-inositol 1,4,5-
Mignen O and Shuttleworth TJ (2000) IARC, a novel arachidonate-regulated, trisphosphate in isolated pancreatic islets. Journal of Biological Chemistry 261:
noncapacitative Ca2þ entry channel. Journal of Biological Chemistry 3501–3511.
275: 9114–9119. Yeung-Yam-Wah V, Lee AK, Tse FW, and Tse A (2010) Arachidonic acid stimulates
Mignen O, Thompson JL, and Shuttleworth TJ (2003) Ca2þ selectivity and fatty acid extracellular Ca2þ entry in rat pancreatic beta cells via activation of the
specificity of the noncapacitative, arachidonate-regulated Ca2þ (ARC) channels. noncapacitative arachidonate-regulated Ca2þ (ARC) channels. Cell Calcium
Journal of Biological Chemistry 278: 10174–10181. 47: 77–83.
 ATP in Plant Mitochondria: Substrates, Inhibitors, and Uncouplers
K L Soole and R I Menz, Flinders University, Adelaide, SA, Australia
ã 2013 Elsevier Inc. All rights reserved.

Glossary Nonphosphorylating reduced nicotinamide

Alternative oxidase An ubiquinol oxidase located in the adenine dinucleotide phosphate (NAD(P)H)
inner mitochondrial membrane, which accepts electrons dehydrogenase(s) Enzymes that accept electrons from
from reduced ubiquinone and reduces oxygen to water. NADH or NADPH and reduce ubiquinone, a mobile
During this process, no protons are translocated across the lipophilic electron carrier in the inner mitochondrial
inner mitochondrial membrane. membrane with no consequent proton translocation across
Nonphosphorylating pathway A route of electron the membrane.
transfer with no concomitant translocation of protons Proton-motive force Refers to the proton gradient that
across the inner mitochondrial membrane; hence, this route is established across the inner mitochondrial membrane
of electron flow does not contribute to the proton- during electron transfer through complexes I, II, and IV,
motive force. often referred to as DmHþ or pmf.

Adenosine triphosphate (ATP) is the energy carrier of the cell turn of the citric acid cycle, one ATP is produced directly by
and in eukaryotic cells is synthesized via photosynthesis and substrate-level phosphorylation, not guanosine triphosphate
respiration. Within respiration, there is a low level of ATP (GTP) as in mammalian mitochondria. Matrix NADH is oxi-
synthesis associated with glycolysis in the cytoplasm; however, dized by the ETC and more ATP is produced via oxidative
the majority of ATP is synthesized via oxidative phosphoryla- phosphorylation.
tion which occurs within mitochondria, specifically via the Oxidative phosphorylation occurs via the interaction of
operation of an electron transport chain (ETC) in the inner large lipoprotein complexes of the ETC and smaller mobile
mitochondrial membrane. In mammals, flux through the respi- electron carriers found in the inner mitochondrial membrane
ratory pathway is tightly regulated by the ATP/adenosine (Figure 1). NADH is oxidized by complex I, which donates its
diphosphate (ADP) ratio or adenylate energy charge of the electrons to a mobile lipophilic electron carrier, ubiquinone.
cell. Plants, unlike mammals, must synthesize all of their cellu- Complex II or succinate dehydrogenase also donates electrons
lar components. In plants, the presence of a nonphosphorylat- to the ubiquinone pool. Reduced ubiquinone (or ubiquinol) is
ing pathway in the mitochondrial ETC and an uncoupler oxidized by complex III via the Q-cycle and reduces the mobile
protein in the inner membrane overcomes this restriction peripheral protein cytochrome c. By interacting with complex
by adenylate control. In plants, respiration not only is impor- IV (cytochrome oxidase), the electrons carried by cytochrome c
tant for energy production but also has a major role in bio- are donated to the terminal electron acceptor, oxygen. During
synthesis and anabolic reactions, in programmed cell death, these electron-transfer events, protons are translocated from
and, more recently, has been implicated as stress response the matrix side of the inner membrane to the intermembrane
signaling sensor. space at complexes I, III, and IV, thus establishing the
proton-motive force (pmf or DmHþ). If ADP is bound to the
F0F1-ATPase in the inner membrane, then protons pass
Mitochondrial ETC in Plants through this complex and the energy within the pmf is used
to synthesize ATP. Once ATP is synthesized, it is then exported
Sucrose and starch are converted to metabolites that feed into out of the matrix in exchange for another ADP via the adenine
the early steps of glycolysis which occurs in the cytoplasm. nucleotide translocase. Thus, the flow of electrons and, hence,
The end products of glycolysis in plants can be either pyruvate oxygen consumption are tightly under control of cellular ADP
or malate, the latter being formed by the action of phosphoe- levels. This is called acceptor or adenylate control.
nol pyruvate carboxylase and malate dehydrogenase, which In plant mitochondria, additional protein complexes
together bypass pyruvate kinase. Once within the mitochon- are found associated with this ETC. They are distinct from
drial matrix, pyruvate is metabolized by pyruvate dehydroge- complexes I to V, in that they participate in the transfer of
nase and the enzymes of the citric acid cycle. Being a substrate electrons from NADH to oxygen, but do not contribute to
for mitochondrial malate dehydrogenase, malate can either the pmf. These enzymes are the alternative or nonphosphor-
feed into the citric acid cycle or be metabolized to pyruvate ylating reduced nicotinamide adenine dinucleotide phosphate
via nicotinamide adenine dinucleotide (NAD)-malic enzyme. (NAD(P)H) dehydrogenases (NDE and NDI), which donate
There must be a balance between these processes, because of electrons to the ubiquinone pool and the alternative oxidase
the poor forward equilibrium of malate dehydrogenase, the (AOX), which accepts electrons from reduced ubiquinone
accumulation of oxaloacetate would prevent further malate (Figure 1).
metabolism. The net result of these reactions is the production Evidence for the alternative nonphosphorylating NAD(P)H
of NADH and FADH2 (via succinate dehydrogenase). During a dehydrogenases comes from the ability of isolated plant

141
142 Bioenergetics | ATP in Plant Mitochondria: Substrates, Inhibitors, and Uncouplers

NAD(P)H NADH Cytoplasm

Cyt b Outer membrane

Porin

NDE 1 NDE 2 Intermembrane space

NAD(P)H H+
H+ NADH Complex V
H+ H+
H+ H+

O2
NADH O2 2H2O Complex III 2H2O UCP
α β α
NADH Alternative Complex IV
NDI 1 oxidase
Complex I NAD(P)H
Succinate
NDI 2 ADP + Pi ATP
Complex II

Cyt c Matrix
Ubiquinone

Figure 1 A schematic representation of the routes of NAD(P)H oxidation and the electron transport chain (ETC) of plant mitochondria. NDI refers
to internal matrix-facing nonphosphorylating NAD(P)H dehydrogenase, and NDE refers to external cytosolic nonphosphorylating NAD(P)H
dehydrogenase. UCP refers to the uncoupling protein. Reproduced from Rasmusson AR, Soole KL, and Elthon TE (2004) Alternative NAD(P)H
dehydrogenases in plant mitochondria. Annual Review of Plant Biology 54: 23–39.

mitochondria to oxidize externally supplied NAD(P)H, allow the release of biosynthetic intermediates from the respi-
which cannot pass through the inner membrane. The external ratory pathway, independent of energy charge of the cell. This
cytosolic-facing NAD(P)H dehydrogenase (NDE) feeds directly role of the nonphosphorylating pathway is hypothesized and
into the ubiquinone pool, as external NAD(P)H oxidation has has not been clearly demonstrated in vivo.
an ADP/O ratio equivalent to succinate of less than 2.0. Matrix
NADH oxidation in plants has a component which is insensi-
tive to inhibition by the complex I inhibitor, rotenone. This Substrates for ATP Synthesis
oxidation is catalyzed by internal inner mitochondrial mem-
brane transdehydrogenases (NDI). The rotenone-insensitive The plant mitochondrial ETC can oxidize matrix NADH and
alternative matrix NADH oxidation has an ADP/O ratio also NADPH to a much lesser extent. The source of this NADH is
similar to succinate indicating that NDI also feeds electrons from pyruvate dehydrogenase and the citric acid cycle, and
into the ubiquinone pool, bypassing proton pumping at com- plants have the full complement of these enzymes in addition
plex I. The AOX is a cyanide and antimycin A-resistant ubiqui- to other unique enzymes. The presence of matrix pools of
nol oxidase that catalyzes the reduction of oxygen to water with NADPH is indicative of the mitochondria’s anabolic role.
electrons from ubiquinol and, thus, bypasses the proton NADP-dependent enzymes involved in folate and thymidylate
pumps at complexes III and IV. Sequence analysis of the synthesis such as NADP-dihydrofolate reductase and methy-
genes putatively encoding NADH(P)H dehydrogenases in var- lene tetrahydrofolate dehydrogenase have been found in plant
ious plant species has separated this gene family into three mitochondria and their continued operation requires the turn-
subfamilies, NDA, NDB, and NDC, based on their homology over of the NADPH generated via NDI2. Apart from NAD-
to the yeast and bacterial gene sequences. There is functional dependent isocitrate dehydrogenase, there is also another
evidence that links NDA to NDI activity and the NDB gene NADP-dependent form in the matrix; however, its role in
family to NDE activities. It is as yet unknown whether NDC plant metabolism is not yet clear. Additionally, via the action
encodes an NDI- or NDE-like activity. of a soluble transhydrogenase detected recently in pea leaf
Thus, it is possible to have electron transport/transfer with matrix, the mitochondrial oxidation of any strictly NAD-linked
no ATP synthesis in plant mitochondria. Therefore, due to the substrates such as pyruvate would also be able to produce
bypasses that exist around key regulatory sites in glycolysis, NADPH.
plant respiration can be totally independent of respiratory Another unique enzyme found in plant mitochondria
control, which gives plants great metabolic flexibility. This is glycine decarboxylase, which generates NADH in the matrix,
flexibility, which results in the loss of adenylate control of is part of photorespiratory cycle and is important in photo-
respiration by the energy status of the cell, can theoretically synthetic metabolism. It is accepted that the mitochondrial
Exploring the Variety of Random
Documents with Different Content
 We should have him back
Who told the Winter’s Tale to do it for us.
Tennyson, Prologue of The Princess.

Winterblossom (Mr. Philip), “the man of taste,” on the managing

committee at the Spa.--Sir W. Scott, St. Ronan’s Well (time, George
III.).

Wintersen (The count), brother of Baron Steinfort, lord of the

place, and greatly beloved.
The Countess Wintersen, wife of the above. She is a kind friend to
Mrs. Haller, and confidante of her brother, the Baron Steinfort.--
Benjamin Thompson, The Stranger (1797).

Winterton (Adam), the garrulous old steward of Sir Edward

Mortimer, in whose service he had been for forty-nine years. He was
fond of his little jokes, and not less so of his little nips, but he loved
his master and almost idolized him.--G. Colman, The Iron Chest
(1796).

Win-the-Fight (Joachin), the attorney employed by Major

Bridgenorth, the roundhead.--Sir W. Scott, Peveril of the Peak (time,
Charles II.).

Winthrop (Madam). One of the oddest chapters in a bona fide

courtship is found in the diary of Judge Samuel Sewall, wherein he
sets down in order the several stages of his wooing of Madame
Winthrop. One extract must suffice.
“I think I repeated again that I would go home and bewail my rashness in
making more haste than good speed. I would endeavor to contain myself and not
go on to solicit her to do that which she could not consent to. Took leave of her.
As came down the steps, she bid me have a care. Treated me courteously. Told
her she had entered the fourth year of her widowhood. I had given her the
newsletter before. I did not bid her draw off her glove as sometime I had done.
Her dress was not so clean as sometime it had been. Jehovah jireh!”--Sewall
Papers (173——).
 Wisdom (Honor paid to).
Anacharsis went from Scythia to Athens to see Solon.--Ælian, De
Varia Historia, v.
Apollonios Tyanæus (Cappadocia) travelled through Scythia and into
India as far as the river Phison to see Hierarchus.--Philostrătos, Life
of Apollonios.
Ben Jonson, in 1619, travelled on foot from London to Scotland
merely to see W. Drummond, the Scotch poet, whose genius he
admired.
Livy went from the confines of Spain to Rome to hold converse
with the learned men of that city.--Pliny the Younger, Epistle, iii 2.
Plato travelled from Athens to Egypt to see the wise men or magi,
and to visit Archytas of Tarentum, inventor of several automatons, as
the flying pigeon, and of numerous mechanical instruments, as the
screw and crane.
Pythagoras went from Italy to Egypt to visit the vaticinators of
Memphis.--Porphyry, Life of Pythagoras.
Sheba (The queen of) went from “the uttermost parts of the earth”
to hear and see Solomon, whose wisdom and greatness had reached
her ear.

Wisdom Persecuted.
Anaxagoras of Clazomēnæ held opinions in natural science so far in
advance of his age that he was accused of impiety, cast into prison,
and condemned to death. It was with great difficulty that Perĭclês
got the sentence commuted to fine and banishment.
Averrois, the Arabian philosopher, was denounced as a heretic, and
degraded, in the twelfth Christian century (died 1226).
Bacon (Friar) was excommunicated and imprisoned for diabolical
knowledge, chiefly on account of his chemical researches (1214-
1294).
Bruno (Giordano) was burnt alive for maintaining that matter is the
mother of all things (1550-1600).
Crosse (Andrew), electrician, was shunned as a profane man,
because he asserted that certain minute animals of the genus Acarus
had been developed by him out of inorganic elements (1784-1855).
 Dee (Dr. John) had his house broken into by a mob, and all his
valuable library, museum, and mathematical instruments destroyed,
because he was so wise that “he must have been allied with the
devil” (1527-1608).
Feargil. (See “Virgilius.”)
Galileo was imprisoned by the Inquisition for daring to believe that
the earth moved round the sun and not the sun round the earth. In
order to get his liberty, he was obliged to “abjure the heresy;” but as
the door closed he muttered, E pur si muove (“But it does move,
though”), (1564-1642).
Gerbert, who introduced algebra into Christendom, was accused of
dealing in the black arts, and was shunned as a “son of Belial.”
Grosted or Grosseteste, bishop of Lincoln, author of some two
hundred works, was accused of dealing in the black arts, and the
pope wrote a letter to Henry III., enjoining him to disinter the bones
of the too-wise bishop, as they polluted the very dust of God’s acre
(died 1253).
Faust (Dr.), the German philosopher, was accused of diabolism for
his wisdom so far in advance of the age.
Peyrere was imprisoned in Brussels for attempting to prove that
man existed before Adam (seventeenth century).
Protagoras, the philosopher, was banished from Athens, for his
book On the Gods.
Socratés was condemned to death as an atheist, because his
wisdom was not in accordance with that of the age.
Virgilius, bishop of Saltzburg, was compelled by Pope Zachary to
retract his assertion that there are other “worlds” besides our earth,
and other suns and moons besides those which belong to our
system (died 784).
Geologists had the same battle to fight, and so had Colenso,
bishop of Natal.

Wise (The).
Albert II., duke of Austria, “The Lame and Wise” (1289, 1330-
1358).
Alfonso X. of Leon and Castile (1203, 1252-1284).
 Charles V. of France, Le Sage (1337, 1364-1380).
Che-Tsou of China (*, 1278-1295).
Comte de las Casas, Le Sage (1766-1842).
Frederick, elector of Saxony (1463, 1544-1554).
James I., the “Solomon” of England (1566, 1603-1625).
John V., duke of Brittany, “The Good and Wise” (1389, 1399-
1442).

Wise Men (The Seven): (1) Solon of Athens, (2) Chilo of Sparta,
(3) Thalês of Milētos, (4) Bias of Priēnê, (5) Cleobūlos of Lindos, (6)
Pittăcos of Mitylēnê, (7) Periander of Corinth, or, according to Plato,
Myson of Chenæ. All flourished in the sixth century B.C.

First Solon, who made the Athenian laws;

While Chilo, in Sparta, was famed for his saws;
In Milētos did Thales astronomy teach;
Bias used in Priēnê his morals to preach;
Cleobulos, of Lindos, was handsome and wise;
Mitylenê ’gainst thraldom saw Pittacos rise;
Periander is said to have gained, thro’ his court,
The title that Myson, the Chenian, ought.

One of the chapters in Plutarch’s Moralia is entitled, “The Banquet

of the Seven Wise Men,” in which Periander is made to give an
account of a contest at Chalcis between Homer and Hesiod. The
latter won the prize, and caused this inscription to be engraved on
the tripod presented to him:

This Hesiod vows to the Heliconian nine,

In Chalcis won from Homer the divine.

Wise Men of the East. Klopstock, in The Messiah, v., says there
were six “Wise Men of the East,” who, guided by the star, brought
their gifts to Jesus, “the heavenly babe,” viz., Ha´dad, Selima, Zimri,
Mirja, Be´led and Sun´ith. (See Cologne, Three Kings of.)
 Wisest Man. So the Delphic oracle pronounced Soc´ratês to be.
Socratês modestly made answer, ’Twas because he alone had learnt
this first element of truth, that he knew nothing.

Not those seven sages might him parallel;

For he whom Pythian maid did whilome tell
To be the wisest man that then on earth did dwell.
Phin. Fletcher, The Purple Island, vi. (1633).

Wisheart (The Rev. Dr.), chaplain to the earl of Montrose.--Sir W.

Scott, Legend of Montrose (time, Charles I.).

Wishfort (Lady), widow of Sir Jonathan Wishfort; an irritable,

impatient, decayed beauty, who painted and enamelled her face to
make herself look blooming, and was afraid to frown lest the enamel
might crack. She pretended to be coy, and assumed, at the age of
60, the airs of a girl of 16. A trick was played upon her by Edward
Mirabell, who induced his lackey, Waitwell, to personate Sir Rowland,
and make love to her; but the deceit was discovered before much
mischief was done. Her pet expression was, “As I’m a person.”--W.
Congreve, The Way of the World (1700).

Wishing-Cap (The), a cap given to Fortunatus. He had only to

put the cap on and wish, and whatever he wished he instantly
obtained.--Straparola, Fortunatus.

Wishing-Rod (The), a rod of pure gold, belonging to the

Nibelungs. Whoever possessed it could have anything he desired to
have, and hold the whole world in subjection.--The Nibelungen Lied,
1160 (1210).

Wishing-Sack (The), a sack given by our Lord to a man named

“Fourteen,” because he was as strong as fourteen men. Whatever he
wished to have he had only to say, “Come into my sack,” and it came
in.
⁂ This is a Basque legend. In Gascoigne it is called Le Sac de la
Ramée (“Ramée’s Sack”).
 Wit--Simplicity. It was said of John Gay that he was

In wit a man, simplicity a child.

⁂ The line is often flung at Oliver Goldsmith, to whom, indeed, it

equally applies.

Witch. The last person prosecuted before the lords or justiciary

(in Scotland) for witchcraft was Elspeth Rule. She was tried May 3,
1709, before Lord Anstruther, and condemned to be burned on the
cheek, and banished from Scotland for life.--Arnot, History of
Edinburgh, 366, 367.

Witch-Finder, Matthew Hopkins (seventeenth century). In 1645

he hanged sixty witches in his own county (Essex) alone, and
received 20s. a head for every witch he could discover.

Has not the present parliament

Mat Hopkins to the devil sent,
Fully empowered to treat about,
Finding revolted witches out?
And has not he within a year
Hanged three score of them in one shire?
S. Butler, Hudibras, ii. 3 (1664).

Witch of Atlas, the title and heroine of one of Shelley’s poems.

Witch of Balwer´y, Margaret Aikens, a Scotchwoman (sixteenth

century).

Witch of Edmonton (The), called “Mother Sawyer.” This is the

true traditional witch; no mystic hag, no weird sister, but only a poor,
deformed old woman, the terror of villagers, and amenable to
justice.
 Why should the envious world
Throw all their scandalous malice upon me?
Because I’m poor, deformed, and ignorant,
And, like a bow, buckled and bent together
By some more strong in mischiefs than myself.
The Witch of Edmonton, (by Rowley, Dekker and Ford,
1658).

Witch’s Blood. Whoever was successful in drawing blood from a

witch, was free from her malignant power. Hence Talbot, when he
sees La Pucelle, exclaims, “Blood will I draw from thee; thou art a
witch!”--Shakespeare, 1 Henry VI. act i. sc. 5 (1592).

Witherington (General), alias Richard Tresham, who first

appears as Mr. Matthew Middlemas.
Mrs. Witherington, wife of the general, alias Mrs. Middlemas (born
Zelia de Monçada). She appears first as Mrs. Middlemas.--Sir W.
Scott, The Surgeon’s Daughter (time, George II.).

Wititterly (Mr. Henry), an important gentleman, 38 years of age;

of rather plebeian countenance, and with very light hair. He boasts
everlastingly of his grand friends. To shake hands with a lord was a
thing to talk of, but to entertain one was to be in the seventh
heaven.
Mrs. Wititterly [Julia], wife of Mr. Wititterly, of Cadŏgan Place,
Sloane Street, London; a faded lady living in a faded house. She
calls her page Alphonse (2 syl.), “although he has the face and
figure of Bill.” Mrs. Wititterly toadies the aristocracy, and, like her
husband, boasts of her grand connections and friends.--C. Dickens,
Nicholas Nickleby (1838). (See Tibbs).

Witling of Terror, Bertrand Barère; also called “The Anacreon of

the Guillotine” (1755-1841).

Wittenbold, a Dutch commandant in the service of Charles II.--

Sir W. Scott, Old Mortality (time, Charles II.).
 Witterington (Roger). (See Widdrington.)

Wittol (Sir Joseph), an ignorant, foolish simpleton, who says that

Bully Buff “is as brave a fellow as Cannibal.”--Congreve, The Old
Bachelor (1693).

Witwould (Sir Wilful), of Shropshire, half-brother of Anthony

Witwould, and nephew of Lady Wishfort. A mixture of bashfulness
and obstinacy, but when in his cups as loving as the monster in the
Tempest. He is “a superannuated old bachelor,” who is willing to
marry Millamant; but as the young lady prefers Edward Mirabell, he
is equally willing to resign her to him. His favorite phrase is, “Wilful
will do it.”
Anthony Witwould, half-brother to Sir Wilful. “He has good nature
and does not want wit.” Having a good memory, he has a store of
other folks’ wit, which he brings out in conversation with good
effect.--W. Congreve, The Way of the World (1700).

Wives as they Were and Maids as they Are, a comedy by

Mrs. Inchbald (1797). Lady Priory is the type of the former, and Miss
Dorrillon of the latter. Lady Priory is discreet, domestic, and
submissive to her husband; but Miss Dorrillon is gay, flighty, and
fond of pleasure. Lady Priory, under false pretences, is allured from
home by a Mr. Bronzely, a man of no principle and a rake; but her
quiet, innocent conduct quite disarms him, and he takes her back to
her husband, ashamed of himself, and resolves to amend. Miss
Dorrillon is so involved in debt that she is arrested, but her father
from the Indies pays her debts. She also repents, and becomes the
wife of Sir George Evelyn.

Wives of Literary Men. According to popular rumor the

following were unhappy in their wives:--Addison, Byron, Dickens,
Dryden, Albert Dürer, Hooker, Ben Jonson, W. Lilly, Milton (first wife),
Molière, More, Saadi, Scaliger, Shakespeare, Shelley, Socratês,
Wycherly, etc. The following were happy in their choice:--Thomas
Moore, Sir W. Scott, Wordsworth, William Howitt, Robert Browning,
S. C. Hall, Disraeli, Gladstone, etc., in England, and in America a
great majority of literary men:--Longfellow, Lowell, Emerson,
Hawthorne, to name only a few.

Wizard of the North, Sir Walter Scott (1771-1832).

Wobbler (Mr.), of the Circumlocution Office. When Mr. Clennam,

by the direction of Mr. Barnacle, in another department of the office,
called on this gentleman, he was telling a brother clerk about a rat-
hunt, and kept Clennam waiting a considerable time. When at length
Mr. Wobbler chose to attend, he politely said, “Hallo, there! What’s
the matter?” Mr. Clennam briefly stated his question; and Mr.
Wobbler replied, “Can’t inform you. Never heard of it. Nothing at all
to do with it. Try Mr. Clive.” When Clennam left, Mr. Wobbler called
out, “Mister! Hallo, there! Shut the door after you. There’s a devil of
a draught!”--Charles Dickens, Little Dorrit, x. (1857).

Woeful Countenance (Knight of the). Don Quixote was so called

by Sancho Panza, but after his adventure with the lions he called
himself “The Knight of the Lions.”--Cervantes, Don Quixote, I. iii. 5;
II. i. 17 (1605-15).

Wolf. The Neuri, according to Herodŏtus, had the power of

assuming the shape of wolves once a year.
One of the family of Antæus, according to Pliny, was chosen
annually, by lot, to be transformed into a wolf, in which shape he
continued for nine years.
Lyca´on, king of Arcādia, was turned into a wolf because he
attempted to test the divinity of Jupiter by serving up to him “human
flesh at table.”--Ovid.
Veret´icus, king of Wales, was turned by St. Patrick into a wolf.

Wolf. When Dantê, in the first Canto of the Divina Commedia,

describes the ascent of the hill (of fame?) he is met, first by a
panther (pleasure?) then by a lion (ambition?) then by a she-wolf
(avarice?)
 A she-wolf, ... who in her leanness seemed
Full of all wants, ... with such fear
O’erwhelmed me ... that of the height all hope I lost.
Dantê, Inferno, i. (1300).

Wolf (To cry), to give a false alarm.

Yöw-wâng, emperor of China, was greatly enamoured of a
courtezan named Pao-tse, whom he tried, by sundry expedients, to
make laugh. At length he hit upon the following plan:--He caused
the tocsins to be rung, the drums to be beaten, and the signal-fires
to be lighted, as if some invader was at the gates. Pao-tse was
delighted, and laughed immoderately to see the vassals and
feudatory princes pouring into the city, and all the people in
consternation. The emperor, pleased with the success of his trick,
amused his favorite over and over again by repeating it. At length an
enemy really did come, but when the alarm was given no one
heeded it, and the emperor was slain (B.C. 770).

Wolf duke of Gascony, one of Charlemagne’s paladins. He was

the originator of the plan of tying wetted ropes round the temples of
his prisoners, to make their eye-balls start from their sockets. It was
he also who had men sewn up in freshly stripped bulls’ hides, and
exposed to the sun till the hides, in shrinking, crushed their bones.--
L’Epine, Croquemitaine, iii.

Wolf of France (She-), Isabella la Belle, wife of Edward II. She

murdered her royal husband “by tearing out his bowels with her own
hands.”

She-wolf of France, with unrelenting fangs,

Thou tear’st the bowels of thy mangled mate.
Gray, The Bard (1757).

Wol´fort, usurper of the earldom of Flanders.--Beaumont and

Fletcher, The Beggars’ Bush (1622).
 Wolfort Webber, Old Knickerbocker, searcher for treasure buried
by buccaneers.--Washington Irving, Tales of a Traveller.

Wolsey (Cardinal), introduced by Shakespeare in his historic play

of Henry VIII. (1601).

Woman Changed to a Man. Iphis, daughter of Lygdus and

Telethusa, of Crete. The story is that the father gave orders if the
child about to be born proved to be a girl, it was to be put to death;
and that the mother, unwilling to lose her infant, brought it up as a
boy. In due time the father betrothed his child to Ianthê, and the
mother, in terror, prayed for help, when Isis, on the day of marriage,
changed Iphis to a man.--Ovid, Metaph. ix. 12; xiv, 699.
Cæneus [Se.nuce], was born of the female sex, but Neptune
changed her into a man. Ænēas, however, found her in the infernal
regions restored to her original sex.
Tire´sias, was converted into a woman for killing one of two
serpents he met in a wood and was restored to his original sex by
killing the other serpent met again after seven years.
D’Eon de Beaumont, the Chevalier, was believed to be a woman.
Hermaphroditos was of both sexes.

Woman killed with Kindness (A), a tragedy by Thos. Heywood

(1600). The “woman” was Mrs. Frankford, who was unfaithful to her
marriage vow. Her husband sent her to live on one of his estates,
and made her a liberal allowance; she died, but on her death-bed
her husband came to see her, and forgave her.

Woman made of Flowers. Gwydion, son of Don, “formed a

woman out of flowers,” according to the Bard Taliesin. Arianrod had
said that Llew Llaw Gyffes (i.e., “The Lion with the Steady Hand”),
should never have a wife of the human race. So Math and Gwydion,
two enchanters,
Took blossoms of oak, and blossoms of broom, and blossoms of meadow-sweet,
and produced therefrom a maiden, the fairest and most graceful ever seen, and
baptized her Blodeuwedd, and she became his bride.--The Mabinogion (“Math,”
etc., twelfth century).

Woman’s Wit, or Love’s Disguises, a drama by S. Knowles

(1838). Hero Sutton loved Sir Valentine de Grey, but offended him by
waltzing with Lord Athunree. To win him back she assumed the
disguise of a Quakeress, called herself Ruth, and pretended to be
Hero’s cousin. Sir Valentine fell in love with Ruth, and then found out
that Ruth and Hero were one and the same person. The secondary
plot is that of Helen and Walsingham, lovers. Walsingham thought
Helen had played the wanton with Lord Athunree, and he
abandoned her. Whereupon Helen assumed the garb of a young
man named Eustace, became friends with Walsingham, said she was
Helen’s brother; but in the brother he discovers Helen herself, and
learnt that he had been wholly misled by appearances.

Women (The Nine Worthy): (1) Minerva, (2) Semiramis, (3)

Tomyris, (4) Jael, (5) Debŏrah, (6) Judith, (7) Britomart, (8)
Elizabeth or Isabella of Aragon, (9) Johanna of Naples.
By’r lady, maist story-man, I am well afraid thou hast done with thy talke. I had
rather have herd something sayd of gentle and meeke women, for it is euill
examples to let them understand of such studye manlye women as those have
been which erewhile thou hast tolde of. They are quicke enow, I warrant you,
noweadays, to take hart-a-grace, and dare make warre with their husbandes. I
would not vor the price o’ my coate, that Jone, my wife had herd this yeare; she
would haue carried away your tales of the nine worthy women a dele zoner than
our minister’s tales anent Sarah, Rebekah, Ruth, and the ministering women, I
warrant you.--John Ferne, Dialogue on Heraldry (“Columel’s reply to Torquatus”).

⁂ “Hart-a-grace,” a hart permitted by royal proclamation to run

free and unharmed for ever, because it has been hunted by a king or
queen.

Women of Abandoned Morals.

Barbara of Cilley, second wife of the Emperor Sigismund, called
“The Messalīna of Germany.”
 Berri (Madame de), wife of the Duc de Berri (youngest grandson
of Louis XIV.).
Catherine II. of Russia, called “The Modern Messalina” (1729-
1796).
Giovanna or Jean of Naples. Her first love was James, count of
March, who was beheaded. Her second was Camicioli, whom she put
to death. Her next was Alfonso of Aragon. Her fourth was Louis
d’Anjou, who died. Her fifth was René, the brother of Louis.
Isabelle of Bavaria, wife of Charles VI., and mistress of the duke of
Burgundy.
Isabelle of France, wife of Edward II., and mistress of Mortimer.
Julia, daughter of the Emperor Augustus.
Marozia, the daughter of Theodora, and mother of Pope John XI.
The infamous daughter of an infamous mother (ninth century.)
Messali´na, the wife of Claudius, the Roman emperor.

Wonder (The), a comedy by Mrs. Centlivre; the second title being

A Woman Keeps a Secret (1714). The woman referred to is Violantê,
and the secret she keeps is that Donna Isabella, the sister of Don
Felix, has taken refuge under her roof. The danger she undergoes in
keeping the secret is this: Her lover, Felix, who knows that Colonel
Briton calls at the house, is jealous, and fancies that he calls to see
Violantê. The reason why Donna Isabella has sought refuge with
Violantê is to escape a marriage with a Dutch gentleman whom she
dislikes. After a great deal of trouble and distress, the secret is
unravelled, and the comedy ends with a double marriage, that of
Violantê with Don Felix, and that of Isabella with Colonel Briton.

Wonder of the World (The).

Gerbert, a man of prodigious learning. When he was made pope,
he took the name of Sylvester II. (930, 999-1003).
Otto III. of Germany, a pupil of Gerbert. What he did deserving to
be called Mirabilia Mundi nobody knows (980, 983-1002).
Frederick II. of Germany (1194, 1215-1250).

Wonderful Doctor, Roger Bacon (1214-1292).

 Wood (Babes in the), a baby boy and girl left by a gentleman of
Norfolk on his death-bed to the care of his brother. The boy was to
have £300 a year on coming of age, and little Jane £500 as a
wedding portion. The uncle promised to take care of the children,
but scarcely had a year gone by when he hired two ruffians to make
away with them. The hirelings took the children on horseback to
Wayland Wood, where they were left to die of cold and hunger. The
children would have been killed, but one of the fellows relented,
expostulated with his companion, and finally slew him. The survivor
compromised with his conscience by leaving the babes alive in the
wood. Everything went ill with the uncle from that hour; his children
died, his cattle died, his barns were set on fire, and he himself died
in jail.
⁂ The prettiest version of this story is one set to a Welsh tune;
but Percy has a version in his Reliques of Ancient English Poetry.

Woodcock (Adam), falconer of the Lady Mary at Avenel Castle.

In the revels he takes the character of the “abbot of Unreason.”--Sir
W. Scott, The Abbot (time, Elizabeth).

Woodcock (Justice), a gouty, rheumatic, crusty, old country

gentleman, who invariably differed with his sister, Deb´orah, in
everything. He was a bit of a Lothario in his young days, and still
retained a somewhat licorous tooth. Justice Woodcock had one child,
named Lucinda, a merry girl, full of frolic and fun.
Deborah Woodcock, sister of the justice; a starch, prudish old
maid, who kept the house of her brother, and disagreed with him in
everything.--Isaac Bickerstaff, Love in a Village (1762).

Woodcocks (The). John Woodcock, a rough, reckless colonist,

who seems harsh to his motherless girl while she is a child, but
subsequently betrays the depths of fatherly affection when she is
persecuted by others.
Mary Woodcock, wild, wayward, passionate girl, in trouble from
her youth up. She marries a gentle-hearted fellow, Hugh Parsons; is
tried for slandering a neighbor, and, driven insane by ill-treatment,
murders her baby, believing it to be a changeling. She is tried for
witchcraft, and acquitted; for child-murder, and sentenced to death,
but dies before the sentence is carried into execution. Her father
says over her lifeless body:
“If I didn’t think the Lord would see just how she’s been abused and knocked
round, and would allow for the way she was brung up, and would strike out all
He’s got agin her, excepting that that didn’t come from bein’ meddled with and
insulted and plagued, I should want to have her an’ me an’ everybody else I care
anything about, blown into a thousand flinders, body and soul, and all the pieces
lost.”--J. G. Holland, The Bay Path (1857).

Woodcourt (Allan), a medical man, who married Esther

Summerson. His mother was a Welsh woman, apt to prose on the
subject of Morgan-ap-Kerrig.--C. Dickens, Bleak House (1852).

Wooden Horse (The). Virgil tells us that Ulysses had a monster

wooden horse, made by Epēos after the death of Hector, and gave
out that it was an offering to the gods to secure a prosperous
voyage back to Greece. By the advice of Sinon, the Trojans dragged
the horse into Troy for a palladium; but at night the Grecian soldiers
concealed therein were released by Sinon from their concealment,
slew the Trojan guards, opened the city gates, and set fire to Troy.
Arctīnos of Milētus, in his poem called The Destruction of Troy,
furnished Virgil with the tale of “the Wooden Horse” and “the
burning of Troy” (fl. B.C. 776).
A remarkable parallel occurred in Saracenic history. Arrestan, in
Syria, was taken in the seventh century by Abu Obeidah by a similar
stratagem. He obtained leave of the governor to deposit in the
citadel some old lumber which impeded his march. Twenty large
boxes filled with men were carried into the castle. Abu marched off;
and, while the Christians were returning thanks for the departure of
the enemy, the soldiers removed the sliding bottoms of the boxes
and made their way out, overpowered the sentries, surprised the
great church, opened the city gates, and Abu, entering with his
army, took the city without further opposition.--Ockley, History of the
Saracens, i. 185 (1718).
 The capture of Sark affords another parallel. Sark was in the
hands of the French. A Netherlander, with one ship, asked
permission to bury one of his crew in the chapel. The French
consented, provided the crew came on shore wholly unarmed. This
was agreed to, but the coffin was full of arms, and the crew soon
equipped themselves, overpowered the French, and took the island.-
-Percy, Anecdotes, 249.

Swoln with hate and ire, their huge, unwieldy force

Came clustering like the Greeks out of the wooden horse.
Drayton, Polyolbion, xii. (1613).

Wooden Horse (The), Clavilēno, the wooden horse on which Don

Quixote and Sancho Panza got astride to disenchant Antonomas´ia
and her husband, who were shut up in the tomb of Queen Maguncia
of Candaya.--Cervantes, Don Quixote, II. iii. 4, 5 (1615).
Another wooden horse was the one given by an Indian to the
shah of Persia as a New Year’s gift. It had two pegs; by turning one
it rose into the air, and by turning the other it descended wherever
the rider wished. Prince Firouz mounted the horse, and it carried him
instantaneously to Bengal.--Arabian Nights (“The Enchanted Horse”).
Reynard says that King Crampart made for the daughter of King
Marcadigês a wooden horse which would go a hundred miles an
hour. His son, Clamadês, mounted it, and it flew out of the window
of the king’s hall, to the terror of the young prince.--Alkman,
Reynard the Fox (1498). (See Cambuscan.)

Wooden Walls, ships made of wood. When Xerxes invaded

Greece, the Greeks sent to ask the Delphic oracle for advice, and
received the following answer (B.C. 480):--

Pallas hath urged, and Zeus, the sire of all,

Hath safety promised in a wooden wall;
Seed-time and harvest, sires shall, weeping, tell
How thousands fought at Salamis, and fell.
 Woodman (The), an opera by Sir H. Bate Dudley (1771). Emily
was the companion of Miss Wilford, and made with Miss Wilford’s
brother “a mutual vow of inviolable affection;” but Wilford’s uncle
and guardian, greatly disapproving of such an alliance, sent the
young man to the Continent, and dismissed the young lady from his
service. Emily went to live with Goodman Fairlop, the woodman, and
there Wilford discovered her in an archery match. The engagement
was renewed, and terminated in marriage. The woodman’s daughter,
Dolly, married Matthew Medley, the factotum of Sir Walter Waring.

Woodstal (Henry), in the guard of Richard Cœur de Lion.--Sir W.

Scott, The Talisman (time, Richard I.).

Woodstock, a novel by Sir W. Scott (1826). It was hastily put

together, but is not unworthy of the name it bears.

Woodville (Harry), the treacherous friend of Penruddock, who

ousted him of the wife to whom he was betrothed. He was wealthy,
but reduced himself to destitution by gambling.
Mrs. Woodville (whose Christian name was Arabella), wife of Harry
Woodville, but previously betrothed to Roderick Penruddock. When
reduced to destitution Penruddock restored to her the settlement
which her husband had lost in play.
Captain Henry Woodville, son of the above; a noble soldier, brave
and high-minded, in love with Emily Tempest, but, in the ruined
condition of the family, unable to marry her. Penruddock makes over
to him all the deeds, bonds and obligations which his father had lost
in gambling.--Cumberland, The Wheel of Fortune (1779).

Woodville (Lord), a friend of General Brown. It was Lord

Woodville’s house that was haunted by the “lady in the Sacque.”--Sir
W. Scott, The Tapestered Chamber (time, George III.).

Woolen. It was Mrs. Oldfield, the actress, who revolted at the

idea of being shrouded in woolen. She insisted on being arrayed in
chintz trimmed with Brussels lace, and on being well rouged to hide
the pallor of death. Pope calls her “Narcissa.”

“Odious! In woolen! ’Twould a saint provoke!”

Were the last words that poor Narcissa spoke.
“No, let a charming chintz and Brussels lace
Wrap my cold limbs and shade my lifeless face;
One would not, sure, be frightful when one’s dead!
And, Betty, give this cheek a little red.”
Pope, Moral Essays, i. (1731).

Wopsle (Mr.), parish clerk. He had a Roman nose, a large,

shining, bald forehead, and a deep voice, of which he was very
proud. “If the Church had been thrown open,” i.e., free to
competition, Mr. Wopsle would have chosen the pulpit. As it was, he
only punished the “Amens” and gave out the psalms; but his face
always indicated the inward thought of “Look at this and look at
that,” meaning the gent in the reading-desk. He turned actor in a
small metropolitan theatre.--C. Dickens, Great Expectations (1860).

Work (Endless), Penelopê’s web; Vortigern’s Tower; washing the

blackamoor white; etc.

Work-room (My).

“Yet the world is thy field, thy garden,

On earth art Thou still at home.
When thou bendest hither thy hallowing eye,
My narrow work-room seems vast and high,
Its dingy ceiling, a rainbow dome--
Stand ever thus at my wide swung door,
And toil will be toil no more.”
Lucy Larcom, Poetical Works (1885).

World (End of the). This ought to have occurred, according to

Cardinal Nicolas de Cusa, in 1704. He demonstrates it thus: The
Deluge happened in the thirty-fourth jubilee of fifty years from the
Creation (A.M. 1700), and therefore the end of the world should
properly occur on the thirty-fourth jubilee of the Christian era, or A.D.
1704. The four grace years are added to compensate for the blunder
of chronologists respecting the first year of grace.
The most popular dates of modern times for the end of the world,
or what is practically the same thing, the Millennium, are the
following:--1757, Swedenborg; 1836, Johann Albrecht Bengel,
Erklärte Offenbarung; 1843, William Miller, of America; 1866, Dr.
John Cumming; 1881, Mother Shipton.
It was very generally believed in France, Germany, etc., that the
end of the world would happen in the thousandth year after Christ;
and therefore much of the land was left uncultivated, and a general
famine ensued. Luckily, it was not agreed whether the thousand
years should date from the birth or the death of Christ, or the
desolation would have been much greater. Many charters begin with
these words, As the world is now drawing to its close. Kings and
nobles gave up their state: Robert of France, son of Hugh Capet,
entered the monastery of St. Denis; and at Limoges, princes, nobles,
and knights proclaimed “God’s Truce,” and solemnly bound
themselves to abstain from feuds, to keep the peace towards each
other, and to help the oppressed.--Hallam, The Middle Ages (1818).
Another hypothesis is this: As one day with God equals a thousand
years (Psalm xc. 4), and God labored in creation six days, therefore
the world is to labor 6000 years, and then to rest. According to this
theory, the end of the world ought to occur A.M. 6000, or A.D. 1996
(supposing the world to have been created 4004 years before the
birth of Christ). This hypothesis, which is widely accepted, is quite
safe for another century at least.

Worldly Wiseman (Mr.), one who tries to persuade Christian

that it is very bad policy to continue his journey towards the Celestial
City. Bunyan, Pilgrim’s Progress, i. (1678).

Worms (Language of). Melampos the prophet was acquainted

with the language of worms, and when thrown into a dungeon,
heard the worms communicating to each other that the roof
overhead would fall in, for the beams were eaten through. He
imparted this intelligence to his jailers, and was removed to another
dungeon. At night the roof did fall, and the king, amazed at this
foreknowledge, released Melampos, and gave him the oxen of
Iphiklos.

Worse than a Crime. Talleyrand said of the murder of the Duc

d’Enghien by Napoleon I. “It was worse than a crime, it was a
blunder.”

Worthies (The Nine). Three Gentiles: Hector, Alexander, Julius

Cæsar; three Jews: Joshua, David, Judas Maccabæus; three
Christians: Arthur, Charlemagne, Godfrey of Bouillon.

Worthies of London. (The Nine).

1. Sir William Walworth, fishmonger, who stabbed Wat Tyler, the
rebel. For this service King Richard II. gave him the “cap of
maintenance” and a “dagger” for the arms of London (Lord Mayor,
1374, 1380).
2. Sir Henry Pritchard or Picard, vintner, who feasted Edward III.,
the Black Prince, John, king of Austria, the king of Cyprus, and David
of Scotland, with 5000 guests, in 1356, the year of his mayoralty.
3. Sir William Sevenoke, grocer. “A foundling, found under seven
oaks.” He fought with the dauphin, and built twenty almshouses, etc.
(Lord Mayor, 1418).
4. Sir Thomas White, merchant tailor, who, during the mayoralty in
1553, kept London faithful to Queen Mary during Wyatt’s rebellion.
Sir Thomas White was the son of a poor clothier, and began trade as
a tailor with £100. He was the founder of St. John’s College, Oxford,
on the spot where two elms grew from one root.
5. Sir John Bonham, mercer, commander of the army which
overcame Solyman the Great, who knighted him on the field after
the victory, and gave him chains of gold, etc.
6. Sir Christopher Croker, vintner, the first to enter Bordeaux, when
it was besieged. Companion and friend of Edward the Black Prince.
 7. Sir John Hawkwood, tailor, knighted by the Black Prince. He is
immortalized in Italian history as Giovanni Acuti Cavaliero. He died in
Padua.
8. Sir Hugh Caverley, silk-weaver, famous for ridding Poland of a
monstrous bear. He died in France.
9. Sir Henry Maleverer, grocer, generally called “Henry of Cornhill,”
a crusader in the reign of Henry IV., and guardian of “Jacob’s Well.”--
R. Johnson, The Nine Worthies of London (1592).

Worthington (Lieutenant), “the poor gentleman;” a disabled

officer and a widower, very poor, “but more proud than poor, and
more honest than proud.” He was for thirty years in the king’s army,
but was discharged on half-pay, being disabled at Gibraltar by a shell
which crushed his arm. His wife was shot in his arms when his
daughter was but three years old. The lieutenant put his name to a
bill for £500; but his friend dying before he had effected his
insurance Worthington became responsible for the entire sum, and if
Sir Robert Bramble had not most generously paid the bill the poor
lieutenant would have been thrown into jail.
Emily Worthington, the lieutenant’s daughter; a lovely, artless,
affectionate girl, with sympathy for every one, and a most amiable
disposition. Sir Charles Cropland tried to buy her, but she rejected
his proposals with scorn, and fell in love with Frederick Bramble, to
whom she was given in marriage.--C. Colman, The Poor Gentleman
(1802).

Worthy, in love with Melinda, who coquets with him for twelve
months, and then marries him.--G. Farquhar, The Recruiting Officer
(1705).

Worthy (Lord), the suitor of Lady Reveller, who was fond of play.
She became weary of gambling, and was united in marriage to Lord
Worthy.--Mrs. Centlivre, The Basset Table (1706).

Wouvermans (The English), Abraham Cooper. One of his best

pieces is “The Battle of Bosworth Field.”
 Richard Cooper is called “The British Poussin.”

Wrangle (Mr. Caleb), a hen-pecked young husband, of oily

tongue and plausible manners, but smarting under the nagging
tongue and willful ways of his fashionable wife.
Mrs. Wrangle, his wife, the daughter of Sir Miles Mowbray. She
was for ever snubbing her young husband, wrangling with him,
morning, noon, and night, and telling him most provokingly “to keep
his temper.” This couple lead a cat-and-dog life: he was sullen, she
quick tempered; he jealous, she open and incautious.--Cumberland,
First Love (1796).

Wrath’s Hole (The), Cornwall. Bolster, a gigantic wrath, wanted

St. Agnes to be his mistress. She told him she would comply when
he filled a small hole, which she pointed out to him, with his blood.
The wrath agreed, not knowing that the hole opened into the sea;
and thus the saint cunningly bled the wrath to death, and then
pushed him over the cliff. The hole is called “The Wrath’s hole” to
this day, and the stones about it are colored with blood-red streaks
all over.--Polwhele, History of Cornwall, i. 176 (1813).

Wray (Enoch), “the village patriarch,” blind, poor, and 100 years
old; but reverenced for his meekness, resignation, wisdom, piety,
and experience.--Crabbe, The Village Patriarch (1783).

Wrayburn (Eugene), barrister-at-law; an indolent, idle, moody,

whimsical young man, who loves Lizzie Hexam. After he is nearly
killed by Bradley Headstone, he reforms, and marries Lizzie, who
saved his life.--C. Dickens, Our Mutual Friend (1864).

Wren (Jenny), whose real name was Fanny Cleaver, a doll’s

dressmaker, and the friend of Lizzie Hexam, who at one time lodged
with her. Jenny was a little, deformed girl, with a sharp, shrewd face,
and beautiful golden hair. She supported herself and her drunken
father, whom she reproved as a mother might reprove a child. “Oh,”
she cried to him, pointing her little finger, “you bad, old boy! Oh, you
naughty, wicked creature! What do you mean by it?”--C. Dickens,
Our Mutual Friend (1864).

Wrong (All in the), a comedy by A. Murphy (1761). The principal

characters are Sir John and Lady Restless, Sir William Bellmont and
his son, George, Beverley and his sister, Clarissa, Blandford and his
daughter, Belinda. Sir John and Lady Restless were wrong in
suspecting each other of infidelity, but this misunderstanding made
their lives wretched. Beverley was deeply in love with Belinda, and
was wrong in his jealousy of her, but Belinda was also wrong in not
vindicating herself. She knew that she was innocent, and felt that
Beverley ought to trust her, but she gave herself and him needless
torment by permitting a misconception to remain which she might
have most easily removed. The old men were also wrong: Blandford
in promising his daughter in marriage to Sir William Bellmont’s son,
seeing she loved Beverley; and Sir William, in accepting the promise,
seeing his son was plighted to Clarissa. A still further complication of
wrong occurs. Sir John wrongs Beverley in believing him to be
intriguing with his wife; and Lady Restless wrongs Belinda in
supposing that she coquets with her husband; both were pure
mistakes, all were in the wrong, but all in the end were set right.

Wronghead (Sir Francis), of Bumper Hall, and M.P. for

Guzzledown; a country squire, who comes to town for the season,
with his wife, son, and eldest daughter. Sir Francis attends the
House, but gives his vote on the wrong side; and he spends his
money on the hope of obtaining a place under Government. His wife
spends about £100 a day on objects of no use. His son is on the
point of marrying the “cast mistress” of a swindler, and his daughter
of marrying a forger; but Manly interferes to prevent these fatal
steps, and Sir Francis returns home to prevent utter ruin.
Lady Wronghead, wife of Sir Francis; a country dame, who comes
to London, where she squanders money on worthless objects, and
expects to get into “society.” Happily, she is persuaded by Manly to
return home before the affairs of her husband are wholly desperate.
 Squire Richard [Wronghead], eldest son of Sir Francis, a country
bumpkin.
Miss Jenny [Wronghead], eldest daughter of Sir Francis; a silly
girl, who thinks it would be a fine thing to be called a “countess,”
and therefore becomes the dupe of one Basset, a swindler, who calls
himself a “count.”--Vanbrugh and Cibber, The Provoked Husband
(1726).

Wyat. Henry Wyat was imprisoned by Richard III., and when

almost starved a cat appeared at the window-grating and dropped a
dove into his hand. This occurred day after day, and Wyat induced
the warder to cook for him the doves thus wonderfully obtained.
Elijah, the Tishbite, while he lay hidden at the brook Cherith, was
fed by ravens, who brought “bread and flesh” every morning and
evening.--1 Kings xvii. 6.

Wylie (Andrew), ex-clerk of bailie Nicol Jarvie.--Sir W. Scott, Rob

Roy (time, George I.).

Wylie (Joe), mate of the Proserpine, hired by Arthur Wardlaw to

scuttle that vessel, that the insurance-money may be used to
conceal the fact of Wardlaw’s defalcations.--Charles Reade, Foul Play.

Wynebgwrthucher, the shield of King Arthur.--The Mabinogion

(“Kilhwch and Olwen,” twelfth century).

Wynkyn de Worde, the second printer in London (from 1491-

1534). The first was Caxton (from 1476-1491). Wynkyn de Worde
assisted Caxton in the new art of printing.

Wynken.
 Wynken, Blynken and Nod one night,
Sailed off in a wooden shoe--
Sailed on a river of misty light
Into a sea of dew.
“Where are you going, and what do you wish?”
The old moon asked the three.
“We have come to fish for the herring-fish
That live in this beautiful sea,
Nets of silver and gold have we,”
Said Wynken, Blynken and Nod.
Eugene Field, A Little Book of Western Verse, (1889).

Wyo´ming, in Pennsylvania, purchased by an American company

from the Delaware Indians. It was settled by an American colony,
but being subject to constant attacks from the savages the colony
armed in self-defence. In 1778 most of the able-bodied men were
called to join the army of Washington, and in the summer of that
year an army of British and Indian allies, led by Colonel Butler,
attacked the settlement, massacred the inhabitants, and burnt their
houses to the ground.
⁂ Campbell has made this the subject of a poem entitled
Gertrude of Wyoming, but he pronounces the name Wy´oming, and
makes Brant, instead of Butler, the leader of the attack.

Wyvill (William de), a steward of the field at the tournament.--Sir

W. Scott, Ivanhoe (time, Richard I.).
 an´adu, a city mentioned by Coleridge in his fragment,
Kubla Khan. The idea of this poem is borrowed from the
Pilgrimage by Purchas (1613), where Xanadu is called
“Xaindu.” Coleridge says that he composed the poem in a
dream, after reading Purchas’ Pilgrimage.

Xanthos, the horse of Achillês. He spoke with a human voice, like

Balaam’s ass, Adrastos’s horse (Arīon), Fortunio’s horse (Comrade),
Mahomet’s “horse” (Al Borak), Sâleh’s camel, the dog of the seven
sleepers (Katmîr), the black pigeons of Dodona and Ammon, the
king of serpents (Temliha), the serpent which was cursed for
tempting Eve, the talking-bird called bulbul-hēzar, the little green
bird of Princess Fairstar, the White Cat cum quibusdam aliis.

The mournful Xanthus (says the bard of old)

Of Peleus’ warlike son the fortune told.
Peter Pindar [Dr. Wolcott], The Lousiad, v. (1809).

Xantippe (3 syl.), wife of Socrătês; proverbial for a scolding,

nagging, peevish wife. One day, after storming at the philosopher,
she emptied a vessel of dirty water on his head, whereupon Socratês
simply remarked, “Aye, aye, we always look for rain after thunder.”

Xantippe (3 syl.), daughter of Cimo´nos. She preserved the life of

her old father in prison by suckling him. The guard marvelled that
the old man held out so long, and, watching for the solution,
discovered the fact.
Euphra´sia, daughter of Evander, preserved her aged father while
in prison in a similar manner. (See Grecian Daughter.)
 Xavier (François), Florentine priest, son of a cameo cutter, who
finds on the shore of Lake Superior an uncut onyx stone, called by
Black Beaver, the Indian owner, “the devil-stone.” Black Beaver will
not sell it, but his daughter, Marie, in love with Xavier, persuades him
to offer it to the Virgin. Xavier engraves upon it an exquisite
representation of Venus rising from the sea. Black Beaver, seeing his
daughter pining for love of Xavier, offers her to the chief priest of the
mission as Xavier’s wife, and learns that Romish priests cannot
marry. He drinks heavily all night, and the next day departs on a
journey “for stores.” That evening Marie, kneeling at prayer, sees
that the cameo has disappeared from the Virgin’s breast. Next day
François Xavier is found dead in the forest, an arrow in his heart.
When the shaft is withdrawn, the arrow-head remains in his bosom.
A century later, within the skeleton of a man exhumed near Starved
Rock, Illinois, is found a rarely beautiful cameo. “The uncanny thing
rattled within the white ribs.”--Elizabeth W. Champney, The
Heartbreak Cameo.

Xavier de Belsunce (H. François), immortalized by his self-

devotion in administering to the plague-stricken at Marseilles (1720-
22).
⁂ Other similar examples are Charles Borro´meo, cardinal and
archbishop of Milan (1538-1584). St. Roche, who died in 1327 from
the plague caught by him in his indefatigable labors in ministering to
the plague-stricken at Piacenza. Mompesson was equally devoted to
the people of Eyam. Sir John Lawrence, lord mayor of London, is
less known, but ought to be held in equal honor, for supporting
40,000 dismissed servants in the great plague.

Xenoc´rates (4 syl.), a Greek philosopher. The courtezan Laïs

made a heavy bet that she would allure him from his philosophy; but
after she had tried all her arts on him without success, she
exclaimed, “I thought he had been a living man, and not a mere
stone.”
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