The role of an absolutely conserved tryptophan residue in octamer formation and stability in mitochondrial creatine kinases.

In most organisms, mitochondrial creatine kinase (MtCK) is present as dimers and octamers with the latter predominating under physiological conditions. An absolutely conserved tryptophan residue (Trp-264 in chicken sarcomeric MtCK) appears to play a key role in octamer stability. Recently, it has been shown that the sponge Tethya aurantia, a member of the most ancient group of living multi-cellular animals, expresses an obligate, dimeric MtCK that lacks this absolutely conserved tryptophan residue, instead possessing a tyrosine in this position. In the present study we confirm that the absolutely conserved tryptophan residue is lacking in other sponge MtCKs where it is instead substituted by histidine or asparagine. Site directed mutations of the Trp-264 in expression constructs of chicken sarcomeric MtCK and the octameric MtCK from the marine worm Chaetopterus destabilized the octameric quaternary structure producing only dimers. A Tyr-->Trp mutation in an expression construct of the Tethya MtCK construct failed to produce octamerization; Tyr-->His and Tyr-->Asn mutations also yielded dimers. These results, in conjunction with analysis of homology models of Chaetopterus and Tethya MtCKs, strongly support the view that while the absolutely conserved tryptophan residue is important in octamer stability, octamer formation involves a complex suite of interactions between a variety of residues.

Isolation and sequence analysis of the gene for arginine kinase from the chelicerate arthropod, Limulus polyphemus: insights into catalytically important residues.

The gene for arginine kinase (AK; EC 2.7.3.3) from the horseshoe crab, Limulus polyphemus, was cloned and the complete cDNA sequence was determined. An open reading frame with 1071 nucleotides was detected that encodes a 357 amino-acid protein with a calculated M(r) of 40,238. The coding transcript is flanked by 13 and 512 nucleotides of 5' and 3' untranslated regions, respectively. The deduced amino-acid sequence of Limulus AK displays extensive similarity to other arginine kinases, vertebrate and invertebrate creatine kinases (CK) and a glycocyamine kinase (GK). Consensus AK and consensus CK sequences, as well as a GK sequence, were compared to CK peptide regions containing residues presumed to be important in catalysis and/or located in close proximity to the active site. Our comparisons revealed some inconsistencies with hypothesized roles of particular residues in catalytic function.

Evolution of phosphagen kinase. VI. Isolation, characterization and cDNA-derived amino acid sequence of lombricine kinase from the earthworm Eisenia foetida, and identification of a possible candidate for the guanidine substrate recognition site.

Lombricine kinase (LK) from the body wall muscle of the earthworm Eisenia foetida was purified to homogeneity. The enzyme was shown to be a dimer consisting of 40 kDa subunits. The cDNA-derived amino acid sequence of 370 residues of Eisenia LK was determined. The validity of the sequence was supported by chemical sequencing of internal tryptic peptides. This is the first reported lombricine kinase amino acid sequence. Alignment of Eisenia LK with those of creatine kinases (CKs), arginine kinases (AKs) and glycocyamine kinase (GK) suggested a region displaying remarkable amino acid deletions (referred to GS region), as a possible candidate for guanidine substrate recognition site. A phylogenetic analysis using amino acid sequences of all four phosphagen kinases indicates that CK, GK and LK probably evolved from a common immediate ancestor protein.

Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: a model for studying phosphagen kinases.

Phosphagen kinases catalyze the reversible transfer of a phosphoryl group between guanidino phosphate compounds and ADP, thereby regenerating ATP during bursts of cellular activity. Large quantities of highly pure arginine kinase (EC 2.7.3.3), the phosphagen kinase present in arthropods, have been isolated from E. coli, into which the cDNA for the horseshoe crab enzyme had been cloned. Purification involves size exclusion and anion exchange chromatographies applied in the denatured and refolded states. The recombinant enzyme has been crystallized as a transition state analog complex. Near complete native diffraction data have been collected to 1.86 A resolution. Substitution of a recombinant source for a natural one, improvement in the purification, and data collection at cryo temperatures have all yielded significant improvements in diffraction.

Organization of the gene for an invertebrate mitochondrial creatine kinase: comparisons with genes of higher forms and correlation of exon boundaries with functional domains.

Two major gene duplication events are thought to have taken place in the evolution of creatine kinases (CK) in the vertebrates - (1) the formation of distinct mitochondrial (MiCK) and cytoplasmic forms from the primordial gene and (2) subsequent formation of the sarcomeric (sar-) and ubiquitous (ubi-) isoforms of octameric MiCK and muscle (M) and brain (B) isoforms of dimeric, cytoplasmic CK. The genes of these two CK clades reflect a distant divergence as sar- and ubiMiCK genes consistently have nine protein-coding exons while M- and B-CK genes have seven protein-coding exons; these genes share only one common exon. CKs are also widely distributed in the invertebrates and it has recently been shown that MiCKs evolved well before the divergence of the major metazoan groups. In the present communication, we report the structure and topology of the gene for MiCK from the protostome marine worm Chaetopterus variopedatus. The protein-coding region of the gene for this primitive MiCK spans over 10 kb and consists of eight exons, the last five (E4-E8) have identical boundaries to the corresponding exons of sar- and ubiMiCK genes. Exon-3 of the C. variopedatus MiCK gene consists of the corresponding E3 and E4 of the vertebrate MiCKs with no intervening intron. E1 is longer and E2 is shorter in the polychaete MiCK gene than the counterpart sarcomeric and ubiquitous genes. The insertion of the intron in C. variopedatus E3 creating the two exons as well as the rearrangement of the intron between E1 and E2 must have occurred prior to or coincident with the duplication event creating the two vertebrate mitochondrial isoforms. Sarcomeric and ubiMiCKs display substantial differences from their invertebrate MiCK counterparts in properties relating to octamer stability and membrane binding. The evolutionary changes in gene topology may be a component of this functional progression.

Origin of octameric creatine kinases.

Mitochondrial creatine kinase (MiCK) occurs primarily as an octameric form localized in the mitochondrial intermembrane compartment in vertebrate tissues and echinoderm spermatozoa (both deuterostome groups). The octameric quaternary structure is thought to play important functional and enzyme targeting roles. We have found that the spermatozoa of the protostome polychaete Chaetopterus variopedatus contain three distinct isoenzymes of creatine kinase (CK) termed CK1, CK2 and CK3. CK3 appears to be present only in the sperm head/midpiece complex where mitochondria are restricted and has a subunit relative molecular mass (Mr) of 43.4 kDa. Gel permeation chromatography using Superdex 200HR showed that CK3 has a native Mr of 344.9 kDa indicating that this enzyme exists as an octamer. Electron micrographs of negatively stained CK3 preparations show structures which are virtually identical to those that have been seen for octameric vertebrate MiCK. The above observations show that CK3 from C. variopedatus displays great similarities to MiCKs from vertebrates and echinoderms. Octamerization of CK is not an advanced feature. The evolution of octameric subunit association is ancient and occurred prior to the divergence of protostomes and deuterostomes.

A dimeric creatine kinase from a sponge: implications in terms of phosphagen kinase evolution.

This study demonstrates conclusively that tissues of the sponge Tethya aurantia contain significant creatine kinase (CK) activity. This CK was purified and analyzed with respect to a number of physico-chemical properties. Size exclusion chromatography and denaturing gel electrophoresis analyses showed that this enzyme is dimeric. The sequences of several Lys-C endoproteinase peptides from Tethya CK are consistent with this enzyme being a member of the phosphagen kinase family and a true CK. CK in higher organisms exists in a variety of quaternary structure forms--dimer, octamer and large monomer consisting of a three contiguous CK domains. The present results indicate that CK evolved very early in metazoan evolution and that the dimeric structure preceded other subunit association forms.

Expression of horseshoe crab arginine kinase in Escherichia coli and site-directed mutations of the reactive cysteine peptide.

Arginine kinase (AK) from the horseshoe crab Limulus polyphemus was expressed in Escherichia coli. The bulk of expressed protein resided in insoluble inclusion bodies. However, approximately 3 mg enzyme protein/l culture was present as active soluble AK. The AK-containing expression vector construct was subjected to site-directed mutagenesis via a polymerase chain reaction-based megaprimer protocol. The AK reactive cysteine peptide was engineered so that it was identical to the corresponding peptide sequence of creatine kinase, another member of the guanidino kinase enzyme family. The resulting expressed protein had a considerably reduced specific activity but was still specific for arginine/arginine phosphate. No catalytic activity was observed with other guanidine substrates (creatine, glycocyamine, taurocyamine, lombricine). The reactive cysteine peptide, characteristic of all guanidino kinases, very likely plays a minimal role in determining guanidine specificity.

The capacity for the de novo biosynthesis of creatine is present in the tunicate Ciona intestinalis and is likely widespread in other protochordate and invertebrate groups.

Creatine kinase (CK) catalyzes the reversible transfer of thegamma-terminal phosphate of MgATP to the guanidine creatine (Cr) forming MgADP and phosphocreatine (PCr). The CK reaction plays a central role in both temporal and spatial ATP buffering in cells displaying high and variable rates of ATP turnover. There is a constant non-enzymatic conversion of Cr and PCr to creatinine that must be compensated for by biosynthesis and/or dietary uptake. In all true vertebrate craniates, there is a capacity for de novo biosynthesis of Cr as evidenced by the presence of the two enzymes involved in the biosynthetic pathway-arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT). Many protochordates and a broad spectrum of invertebrates, including the basal metazoan sponges, express CK and contain significant pools of Cr/PCr, particularly in primitive-type spermatozoa. However, attempts at demonstrating the enzymes of Cr biosynthesis in these organisms have failed and it has been suggested that Cr is derived from the diet and/or by direct uptake from seawater. We show in this communication that the protochordate tunicate Ciona intestinalis expresses GAMT based on the deduced amino acid sequence of a cDNA amplified by reverse transcription PCR. To validate that the transcript coded for GAMT, a full length cDNA was generated by PCR amplification and ligated into an expression vector. The resulting recombinant protein had an N-terminal amino acid sequence and relative molecular mass as predicted by the deduced amino acid sequence from the cDNA. Catalytic studies of this recombinant GAMT showed that it indeed had the capacity to methylate guanidinocetate to Cr with an apparent K(m) and maximal velocity comparable to GAMTs from vertebrates. Real time PCR showed that this GAMT is primarily expressed in the stomach and gonad, in close proximity to where Cr is packaged into spermatozoa, but also is expressed in two other tissue complexes. Analysis of the C. intestinalis genome and EST sequencing projects showed that the AGAT gene is present and is expressed demonstrating that this species has the complete Cr biosynthetic pathway. Perusal of other EST and genome sequencing projects revealed that true GAMTs are present in the lancelet Branchiostoma, the sea urchin Strongylocentrotus and the hydroid Hydractinia and AGAT genes are present in both Branchiostoma and Strongylocentrotus. Given our present experimental results and the emerging EST/genome sequencing data, it is clear that the capacity for de novo Cr biosynthesis is likely widespread in protochordates and invertebrates expressing CK and that the genes for GAMT/AGAT likely evolved coincident with CK.

Studies of Intracellular pH Regulation in Cardiac Myocytes From the Marine Bivalve Mollusk, Mercenaria campechiensis.

Myocytes were isolated from the ventricle of the marine clam Mercenaria campechiensis by enzymatic dispersion procedures. Intracellular pH (pHi) was measured via fluorescence imaging techniques using an inverted microscope interfaced with a high sensitivity television camera. Myocyte pHi was similar to values observed in other molluscan muscles measured by weak acid distribution and nuclear magnetic resonance (NMR) techniques. Myocytes displayed a good capacity for defending pHi against changes in extracellular pH (pHe) as the pHi remained unchanged in the pHe range of 7.1 to 8.0, but gradually declined at lower pHe values. Myocytes had a relatively high non-bicarbonate intracellular buffering capacity. Further, these cells showed recovery from imposed acid loads. This recovery was accelerated by increasing HCO3- concentrations, was not dependent on external Na+ and was blocked by a stilbene transport inhibitor, suggesting that a HCO3-:CI- transporter plays a central role in regulation of pHi. Collectively, these data show that ventricular myocytes of M. campechiensis have a relatively high capacity for dealing with potential metabolic proton loads associated with environmental anaerobiosis.

Evolution and physiological roles of phosphagen systems.

Phosphagens are phosphorylated guanidino compounds that are linked to energy state and ATP hydrolysis by corresponding phosphagen kinase reactions: phosphagen + MgADP + H(+) <--> guanidine acceptor + MgATP. Eight different phosphagens (and corresponding phosphagen kinases) are found in the animal kingdom distributed along distinct phylogenetic lines. By far, the creatine phosphate/creatine kinase (CP/CK) system, which is found in the vertebrates and is widely distributed throughout the lower chordates and invertebrates, is the most extensively studied phosphagen system. Phosphagen kinase reactions function in temporal ATP buffering, in regulating inorganic phosphate (Pi) levels, which impacts glycogenolysis and proton buffering, and in intracellular energy transport. Phosphagen kinase reactions show differences in thermodynamic poise, and the phosphagens themselves differ in terms of certain physical properties including intrinsic diffusivity. This review evaluates the distribution of phosphagen systems and tissue-specific expression of certain phosphagens in an evolutionary and functional context. The role of phosphagens in regulation of intracellular Pi levels likely evolved early. Thermodynamic poise of the phosphagen kinase reaction profoundly impacts this capacity. Furthermore, it is hypothesized that the capacity for intracellular targeting of CK evolved early as a means of facilitating energy transport in highly polarized cells and was subsequently exploited for temporal ATP buffering and dynamic roles in metabolic regulation in cells displaying high and variable rates of aerobic energy production.

Phosphocreatine represents a thermodynamic and functional improvement over other muscle phosphagens.

In vertebrate tissues, the only phosphagen is phosphocreatine (PC), and the corresponding phosphotransferase is creatine phosphokinase (CPK). Among invertebrates, a variety of phosphotransferase reactions are found in addition to CPK, including arginine phosphokinase (APK), glycocyamine phosphokinase (GPK), taurocyamine phosphokinase (TPK) and lombricine phosphokinase (LPK). Although there is some uncertainty about the exact value, the apparent equilibrium constant for the CPK reaction (K'cpk = [creatine][ATP]/[PC][ADP]), under physiological conditions similar to those of vertebrate muscle, ranges from 100 to 160. The corresponding K' value for the APK reaction is somewhat controversial, and K' values for the GPK. TPK and LPK reactions are not known. In this study, conventional and 31P-NMR methods were used to evaluate the equilibrium constants for the APK, GPK, TPK and LPK reactions relative to that of CPK. The corresponding K' values for the APK, GPK, TPK and LPK reactions, expressed as a percentage of K'cpk, are 13, 29, 29 and 32%, respectively. The exclusively invertebrate phosphagens exist as a cohort of thermodynamically more stable compounds. Thus, PC constitutes a thermodynamic (and functional) improvement, in that the CPK reaction is able to buffer ATP at much higher ATP/ADP ratios than are other phosphagens. However, possession of a phosphagen system with a lower K' value may be advantageous under certain specific physiological conditions such as intracellular acidosis.

Mitochondrial Activities of Phosphagen Kinases are Not Widely Distributed in the Invertebrates.

A diverse array of phosphagen kinases [arginine kinase (AK), lombricine kinase (LK)], glycocyamine kinase (GK), taurocyamine kinase (TK), and creatine kinase (CK) is found in the animal kingdom (see ref. 1 for a review). These reactions appear to function in the temporal buffering of ATP in muscles during energy deficits such as might occur during burst contraction or anoxia (2, 3). In many vertebrate tissues, a distinct mitochondrial isoenzyme of CK is present, and it may play a special role in the intracellular transport of high energy phosphate (4). In this study, we investigated whether mitochondrial activities of phosphagen kinases are present in invertebrate muscles. Our results show that AK is present in mitochondria from a crustacean. However, phosphagen kinases are lacking in mitochondria from insect flight muscles, molluscan cardiac and smooth muscle, and polychaete and oligochaete body wall musculature. It appears that mitochondrial activities of phosphagen kinases are not widely distributed in the invertebrates. These data, in conjunction with previous studies on the physico-chemical nature of the interaction of phosphagen kinases with mitochondria (5, 6), suggest that mitochondrial compartmentation of phosphagen kinases may have evolved independently in two major animal groups.

Graded intracellular acidosis produces extensive and reversible reductions in the effective free energy change of ATP hydrolysis in a molluscan muscle.

Phosphorus nuclear magnetic resonance spectroscopy was used to evaluate the impact of experimental reductions of intracellular pH on in vitro preparations of the radula protractor muscle of the marine gastropod, Busycon canaliculatum. The intracellular pH of radula refractor muscle bundles superfused with buffered artificial sea water (pH = 7.8) was 7.29. It was possible to clamp muscle intracellular pH at various acidotic states by changing the superfusate to 5, 10, and 15 mmol.l-1 5,5-dimethyl-oxazolidine-2,4-dione in buffered artificial sea water (pH = 6.5). Consistent and temporally stable reductions of intracellular pH were achieved (intracellular pH = 6.98, 6.79, and 6.62, respectively). During the acidotic transitions, arginine phosphate concentrations decreased and inorganic phosphate concentrations increased in a reciprocal manner and remained essentially constant after the intracellular pH stabilized. The extent of changes in arginine phosphate and inorganic phosphate was directly proportional to the magnitude of the imposed acidosis. Total adenosine triphosphate concentrations remained unchanged in all treatments. However, the magnesium adenosine triphosphate to total adenosine triphosphate ratio declined in direct relation to the extent of the acidosis. Intracellular free Mg2+ fell incrementally with reduced intracellular pH. All of the above effects were rapidly reversed when the 5,5-dimethyl-oxazolidine-2,4-dione was washed out by changing the superfusate to buffered artificial sea water (pH = 7.8). Mg-adenosine diphosphate concentrations were calculated in all treatments using equilibrium constants for the arginine kinase reaction corrected for pH and intracellular free [Mg2+]. The metabolite, intracellular pH, and [Mg2+] data were used to estimate the effective free energy of hydrolysis of adenosine triphosphate (dG/d xi ATP) under most experimental conditions. Experimental acidosis resulted in dramatic reductions in dG/d xi ATP which were fully reversible upon wash-out of 5,5-dimethyl-dioxazolidine-2,4-dione and recovery to normal intracellular pH conditions. Acidosis resulted in net hydrolysis of arginine phosphate, likely via a complex mechanism involving enhancement of rate of adenosine triphosphate hydrolysis and/or inhibition of adenosine triphosphate synthesis.

Structural and functional implications of the amino acid sequences of dimeric, cytoplasmic and octameric mitochondrial creatine kinases from a protostome invertebrate.

The cDNA and deduced amino-acid sequences for dimeric and octameric isoforms of creatine kinase (CK) from a protostome, the polychaete Chaetopterus variopedatus, were elucidated and then analysed in the context of available vertebrate CK sequences and the recently determined crystal structure of chicken sarcomeric mitochondrial CK (MiCK). As protostomes last shared a common ancestor with vertebrates roughly 700 million years ago, observed conserved residues may serve to confirm or reject contemporary hypotheses about the roles of particular amino acids in functional/structural processes such as dimer/octamer formation and membrane binding. The isolated cDNA from the dimeric CK consisted of 1463 nucleotides with an open reading frame of 1116 nucleotides encoding a 372-amino-acid protein having a calculated molecular mass of 41.85 kDa. The percentage identity of C. variopedatus dimeric CK to vertebrate CK is as high as 69%. The octameric MiCK cDNA is composed of 1703 nucleotides with an open reading frame of 1227 nucleotides. The first 102 nucleotides of the open reading frame encode a 34-amino-acid leader peptide whereas the mature protein is composed of 375 amino acids with a calculated molecular mass of 42.17 kDa. The percentage identity of C. variopedatus MiCK to vertebrate CK is as high as 71%. This similarity is also evident in residues purported to be important in the structure and function of dimeric and octameric CK: (a) presence of seven basic amino acids in the C-terminal end thought to be important in binding of MiCK to membranes; (b) presence of a lysine residue (Lys110 in chicken MiCK) also thought to be involved in membrane binding; and (c) presence of a conserved tryptophan thought to be important in dimer stabilization which is present in all dimeric and octameric guanidino kinases. However, C. variopedatus MiCK lacks the N-terminal heptapeptide present in chicken MiCK, which is thought to mediate octamer stabilization. In contrast with vertebrate MiCK, polychaete octamers are very stable indicating that dimer binding into octamers may be mediated by additional and/or other residues. Phylogenetic analyses showed that both octamer and dimer evolved very early in the CK lineage, well before the divergence of deuterostomes and protostomes. These results indicate that the octamer is a primitive feature of CK rather than being a derived and advanced character.

Functional and Evolutionary Implications of the Distribution of Phosphagens in Primitive-Type Spermatozoa.

External fertilization is considered to be the primitive condition in metazoans. The spermatozoa of such organisms typically display a common primitive-type morphology that is present in a range of phyla. These spermatozoa are extremely polarized cells in that the site of ATP synthesis (mitochondria in midpiece) is located at large diffusion distances from the ATP sink (dynein ATPases in the flagellum). Spermatozoa of polychaetes, sipunculids, echiuroids, echinoderms, and tunicates contain the phosphagen creatine phosphate or express the corresponding phosphagen kinase creatine kinase (or both), even when other phosphagens/phosphagen kinases are present in somatic tissues and eggs. The selective expression of the creatine kinase system in these spermatozoa may be related to potential advantages in the cellular transport of energy. To evaluate this possibility, we compared the efficacy of the major phosphagen systems for cellular transport of energy. We used a facilitated diffusion model for spatial ATP buffering, taking into account relative differences in diffusivity and thermodynamic poise. At low ratios of [total phosphagen pool]/ [total adenine nucleotide pool] (CG+P/CAd ratio), creatine phosphate carried a higher fraction of total high-energy phosphate (J) than the other phosphagens. However, J values for all phosphagens were greater than 0.9, and these differences disappeared as the CG+P/CAd ratio was increased. Thus, the functional benefit of using CP, rather than other phosphagens, in energy transport is quite limited. The creatine kinase system became associated with primitive-type spermatozoa early in metazoan evolution. This association is not necessarily related to inherent advantages of this phosphagen system for buffering of ATP, but may be linked to historical events in the evolution of the cell phenotype.

Gene duplication events producing muscle (M) and brain (B) isoforms of cytoplasmic creatine kinase: cDNA and deduced amino acid sequences from two lower chordates.

Creatine kinase (CK) is coded for by at least four loci in higher vertebrates--two cytoplasmic isoforms, muscle (M) and brain (B), and two mitochondrial isoforms, sarcomeric and ubiquitous. M is expressed primarily in skeletal muscle, while B is expressed in a variety of cells, including cardiac and smooth muscle fibers, neurons, transport epithelia, and photoreceptors. M and B subunits form very stable homodimers (MM [M-CK], BB [B-CK]) and heterodimers (MB). M-CK is capable of binding to the M line of the myofibril, thereby creating an energy transfer microcompartment; BB and MB CKs are not. M- and B-like CKs are present in all vertebrates yet examined, including fish. Cytoplasmic, dimeric CKs are widely distributed in the invertebrates. The only available amino acid sequence for an invertebrate dimeric CK, that of the protostome polychaete Chaetopterus variopedatus, is just as similar to the vertebrate M isoform as to the B isoform. Echinoderms lack dimeric, cytoplasmic CKs, which appear to be replaced by a dimeric arginine kinase which evolved secondarily from CK. Thus, it is likely that the gene duplication event producing the M and B isoforms occurred after the divergence of the chordates from echinoderms. To narrow down the timing of this duplication event, we obtained the cDNA and deduced amino acid sequences of dimeric CKs from the tunicate Ciona intestinalis (subphylum Urochordata) and the lancelet Branchiostoma floridae (subphylum Cephalochordata). Our results show that these CKs are strikingly similar to both invertebrate and vertebrate CKs. However, phylogenetic analyses by neighbor-joining and parsimony show that these two enzymes appeared to have diverged before the point of divergence of the M and B isoforms. Thus, the gene duplication event for formation of the muscle and brain isoforms of CK most likely occurred during the radiation of the fish, a time noted for gene duplication events at a variety of other loci.

Patterns of energy metabolism in the stone crab, Menippe mercenaria, during severe hypoxia and subsequent recovery.

Specimens of the stone crab, Menippe mercenaria, survived severe hypoxia (PO2 less than 8mm Hg) for at least 12 hr at 28-30 degrees C. During the time course of 12 hr of hypoxia, hemolymph L-lactate levels rose to 30-50 mumoles/g wet wt. There was a slight elevation of L-alanine levels, whereas succinate was found in only trace quantities in the hemolymph. Pronounced metabolic changes took place in the heart, cheliped closer, and leg socket muscles during severe hypoxia. L-lactate accumulated to levels ranging from 16-20 mumoles/g wet wt. There were pronounced changes in high-energy phosphate levels in the cheliped closer and leg socket muscles. Taking into account expected intra- and extracellular water content, the calculated intracellular lactate content in the three muscles investigated is substantially less than the hemolymph lactate concentrations. Part of this reverse concentration gradient may be accounted for by the reduction in lactate activity due to cation-lactate complex formation. Hemolymph calcium and magnesium concentrations rose considerably during severe hypoxia. During recovery from severe hypoxia, approximately 50% of the accumulated lactate in the hemolymph was cleared in 6 hr. Hemolymph lactate and alanine levels returned to near control levels after 24 hr of recovery. This study shows that the stone crab, M. mercenaria, survives severe hypoxia by a reliance on glycogen fermentation to lactate. This species is capable of tolerating high levels of accumulated lactate.

The anaerobic molluscan heart: adaptation to environmental anoxia. Comparison with energy metabolism in vertebrate hearts.

The hearts of many bivalve and gastropod molluscs are resistant to exposure to hypoxic and anoxic conditions. Glycogen and aspartate are simultaneously fermented leading to the accumulation of alanine, succinate and alanopine/strombine. Lactate is not a major end product of anaerobic metabolism in molluscan hearts. In contrast, vertebrate hearts respond to hypoxia by the fermentation of glycogen leading to lactate formation. There is some evidence for aspartate and glutamate breakdown in vertebrate hearts during anoxia. However, the quantitative contribution of this process to energy production is small. The differences in modes of energy production in molluscan and vertebrate hearts may reflect adaptations to long-term as opposed to short-term anoxia.