M4 Lactate Dehydrogenase Research Articles

Activation of p53 mediated glycolytic inhibition-oxidative stress-apoptosis pathway in Dalton's lymphoma by a ruthenium (II)-complex containing 4-carboxy N-ethylbenzamide.

There is a general agreement that most of the cancer cells switch over to aerobic glycolysis (Warburg effect) and upregulate antioxidant enzymes to prevent oxidative stress induced apoptosis. Thus, there is an evolving view to target these metabolic alterations by novel anticancer agents to restrict tumor progression in vivo. Previously we have reported that when a non toxic dose (10 mg/kg bw i.p.) of a novel anticancer ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide; Ru(II)-CNEB, was administered to the Dalton's lymphoma (DL) bearing mice, it regressed DL growth by inducing apoptosis in the DL cells. It also inactivated M4-LDH (M4-lactate dehydrogenase), an enzyme that drives anaerobic glycolysis in the tumor cells. In the present study we have investigated whether this compound is able to modulate regulation of glycolytic inhibition-apoptosis pathway in the DL cells in vivo. We observed that Ru(II)-CNEB could decline expression of the inducible form of 6-phosphofructo-2-kinase (iPFK2: PFKFB3), the master regulator of glycolysis in the DL cells. The complex also activated superoxide dismutase (the H2O2 producing enzyme) but declined the levels of catalase and glutathione peroxidase (the two H2O2 degrading enzymes) to impose oxidative stress in the DL cells. This was consistent with the enhanced p53 level, decline in Bcl2/Bax ratio and activation of caspase 9 in those DL cells. The findings suggest that Ru(II)-CNEB is able to activate oxidative stress-apoptosis pathway via p53 (a tumor supressor protein) mediated repression of iPFK2, a key glycolytic regulator, in the DL cells in vivo.

Lactate dehydrogenase from the tetraploid weatherfish Misgurnus fossilis during temperature adaptation: Determination of structural differences in two forms of the enzyme by molecular modeling methods

A structural analysis of two lactate dehydrogenase M4 protein forms has been performed. These structures are the protein products of two lactate dehydrogenase gene (LDH-A) copies in the weatherfish Misgurnus fossilis genome after thermal adaptation (acclimation) to 5 degrees C and 18 degrees C. The localization of three earlier identified amino acid substitutions (Gly214Val, Leu304Ile, Asp312Glu) has been determined, and the molecular dynamics simulation and computer modeling of two forms of the enzyme from skeletal muscles LDH-M4 have been carried out. After molecular dynamics trajectory calculations carried out at 5, 18, and 25 degrees C, the intersubunit distances for all structures used in calculations have been determined. It has been found that the Gly214Val substitution localized in the intersubunit region leads to a new intersubunit interaction, which plays a role in the stabilization of tetrameric enzyme structure after the adaptation to 18 degrees C.

Regression of Dalton’s lymphoma in vivo via decline in lactate dehydrogenase and induction of apoptosis by a ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide as ligand

A novel ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide (Ru(II)-CNEB) was found to interact with and inhibit M4-lactate dehydrogenase (M4-LDH), a tumor growth supportive enzyme, at the tissue level. The present article describes modulation of M4-LDH by this compound in a T-cell lymphoma (Dalton's Lymphoma: DL) vis a vis regression of the tumor in vivo. The compound showed a dose dependent cytotoxicity to DL cells in vitro. When a non toxic dose (10 mg/kg bw i.p.) of Ru(II)-CNEB was administered to DL bearing mice, it also produced a significant decline in DL cell viability in vivo. The DL cells from Ru(II)-CNEB treated DL mice showed a significant decline in the level of M4-LDH with a concomitant release of this protein in the cell free ascitic fluid. A significant increase of nuclear DNA fragmentation in DL cells from Ru(II)-CNEB treated DL mice also coincided with the release of mitochondrial cytochrome c in those DL cells. Importantly, neither blood based biochemical markers of liver damage nor the normal patterns of LDH isozymes in other tissues were affected due to the treatment of DL mice with the compound. These results were also comparable with the effects of cisplatin (an anticancer drug) observed simultaneously on DL mice. The findings suggest that Ru(II)-CNEB is able to regress Dalton's lymphoma in vivo via declining M4-LDH and inducing mitochondrial dysfunction-apoptosis pathway without producing any toxicity to the normal tissues.

Effects of Urea on M4-Lactate Dehydrogenase from Elasmobranchs and Urea-Accumulating Australian Desert Frogs

We measured the effect of urea on M4-lactate dehydrogenase (M4-LDH) from elasmobranchs and Australian desert frogs (urea accumulators) and from two animals that do not accumulate urea, the axolotl and the rabbit. An analysis of the effect of urea on the Kd(NADH), V, V/Km(pyr) and V/Km(NADH) shows that in all cases the major effect of urea was on the binding of pyruvate, which fits with data in the literature that show that urea acts as a competitive inhibitor of LDH. The characteristics of the elasmobranch enzymes are consistent with a proposed adaptation model, but the situation for the enzymes from the aestivating frogs is equivocal. Urea (400 mM) had less effect on the Km(pyr) of M4-LDH from the urea accumulators than it did on the non-accumulators, suggesting a general adaptation and that the enzyme produced by the aestivating frogs (urea accumulators) is kinetically different from that of non-aestivating frogs (non-accumulators). A new approach is used to characterize the overall pattern of adaptation to urea. The pattern is similar in an enzyme from an elasmobranch and an aestivating frog despite the temporary presence of urea in the latter and the phylogenetic difference between these animals.

Proline-Protein Interactions: Protection of Structural and Functional Integrity of M4 Lactate Dehydrogenase

A well defined labile isozyme, rabbit muscle M4 - lactate dehydrogenase was denatured under freeze-thaw, heat and GuHCl treatment in the presence and absence of proline, and the corresponding structural changes of the enzyme were monitored through fluorescence and CD spectral studies. The data reveal that proline confers protection to the structural integrity of the enzyme, thereby protecting its activity. This was attributed to its property of forming hydrophilic colloids in aqueous media with a hydrophobic backbone interacting with protein. Unlike other osmolytes, proline is proposed to act on the enzyme stability not only by inducing preferential hydration of proteins but also through the interactions of its multimeric hydrophobic backbone with the solvent-accessible hydrophobic regions of the enzyme.

Pressure and Lactate Dehydrogenase Function in Diving Mammals and Birds

Emperor penguins (Aptenodytes forster), elephant seals (Mirounga angustirostris), and sperm whales (Physeter catodon) have been shown to dive to considerable depths (265 m, 1,500 m, and 1, 140 m, respectively). These animals must cope with extreme changes in hydrostatic pressure as they dive. The effects of hydrostatic pressure on the Michaelis-Menten constant, $K_{m}$, of cofactor binding of NADH of muscle M₄ (muscle type) lactate dehydrogenase (LDH) was measured for these diving vertebrates and compared with a nondiving mammal, the domestic rabbit. No effect of pressure changes as great as 2.066 X 10⁴ kPa (204 atm) was observed in either the diving or nondiving species LDH preparations. Results support the hypothesis that, at least concerning the $K_{m}$ of NADH in the M₄ LDH of the diving vertebrates examined, the LDHs of warm-blooded divers do not appear to be affected by changes in hydrostatic pressure and the enzyme may be preadapted for insensitivity to pressure perturbations.

Infection of mice with lactate dehydrogenase—elevating virus destroys the subpopulation of kupffer cells involved in receptor-mediated endocytosis of lactate dehydrogenase and other enzymes

In previous experiments in rats, we have shown that the rapid plasma clearance of a number of clinically important enzymes is due to receptor-mediated endocytosis by Kupffer cells and other resident macrophages. Others have shown that infection of mice with lactate dehydrogenase-elevating virus, a virus that proliferates in macrophages, leads to reduced plasma elimination of these enzymes. This paper integrates these two sets of experiments. Plasma elimination of intravenously injected, radioactively labeled lactate dehydrogenase M4 and mitochondrial malate dehydrogenase in mice was shown to be caused in part by uptake in liver, spleen and bone. Uptake of lactate dehydrogenase M4 by these tissues was, to a large extent, saturable and the two dehydrogenases competitively inhibited each other's clearance. These results suggest that, also in mice, these enzymes are partly cleared from plasma by endocytosis by way of a common receptor on cells (probably macrophages) from liver, spleen and bone marrow. Morphometrical data showed that normal mouse liver contains 23 x 10(6) Kupffer cells/cm3. This number was reduced to about 30% of that of controls 24 hr after infection of mice with lactate dehydrogenase-elevating virus but returned to normal within the next 9 days. The saturable component of uptake of lactate dehydrogenase M4 by liver, spleen and bone had disappeared 24 hr after infection with the virus, and did not return after the Kupffer cell population had recovered. Our findings suggest that lactate dehydrogenase M4 is, to a large extent, removed from the circulation by way of a receptor on a subpopulation of macrophages that is permissive for replication of lactate dehydrogenase-elevating virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Pressure-Temperature Interactions on M4-Lactate Dehydrogenases From Hydrothermal Vent Fishes: Evidence for Adaptation to Elevated Temperatures by the Zoarcid Thermarces andersoni, but not by the Bythitid, Bythites hollisi

Lactate dehydrogenases (LDH; M4 isozyme) were purified from skeletal muscle taken from two fishes endemic to hydrothermal vents, Thermarces andersoni (Zoarcidae; 13: °N, East Pacific Rise, depth [sim] 2600 m) and Bythites hollisi (Bythitidae; Galapagos Spreading Center, depth [sim] 2500 m), and from the cosmopolitan deep-sea rattail Coryphaenoides armatus (Macrouridae; depth of occurrence to [sim] 5000 m). The effects of pressure and temperature on the apparent Michaelis-Menten constant (Km) of cofactor (NADH) were measured to compare sensitivities to temperature, at in situ pressures, of enzymes from hydrothermal vent fishes and from a species adapted to cold, stable deep-sea temperatures. At 5: °C, the Km of NADH of the M4-LDHs of the three species varied only slightly between measurement pressures of 1 and 340 atmospheres (atm), in agreement with earlier studies of M4-LDHs of deep-sea fishes. At higher measurement temperatures, marked differences were found among the enzymes. For the M4-LDHs of C. armatus and B. hollisi, increases in temperature (10 to 20: °C), at in situ pressures, sharply increased the Km of NADH to values higher than those predicted to be physiologically optimal. The M4-LDH of T. andersoni exhibited only minimal perturbation by elevated temperature under in situ pressures. The different temperature-pressure responses of these LDHs suggest that enzymes of deep-sea fishes not endemic to hydrothermal vents are not adapted for function at the higher temperatures found at vent sites, and that T. andersoni is better adapted than B. hollisi for sustained exposure to warm vent waters. The importance of adaptation to warm temperatures in the colonization of vent habitats is discussed.

Structure determination and refinment of Bacillus stearothermophilus lactate dehydrogenase

AbstractStructures have been determined of Bacillus stearothermophilus “apo” and holo lactate dehydrogenase. The holo‐enzyme had been co‐crystallized with the activator fructose 1,6‐biosphosphate. The “apo” lactate dehydrogenase structure was solved by use of the known apo‐M4 dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 Å resolution by means of the noncrystallographic molecular 222 symmetry. The R‐factor was reduced to 28.7%, using 2.8 Å resolution data, in a restrained least‐squares refnement in which the molecula rsymmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the “apo” enzyme.Further refinement proceeded with the isomorphous holo‐enzyume from Bacillus Stearothermophilus. After removing the noncrystallographic constraints, the R‐factor dropped from 30.3% to a final value of 26.0% with a 0.019 Å and 1.7° r.m.s. deviation from idealized bond length and angles, respectively.Two sulfate ions per subunit were included in the final model of the “apo” ‐from‐one at the substrate binding site and one close to the molecular P ‐axis near the location of the fructose 1,6‐bisphosphate activator. The final model of the holo‐enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the R‐axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6‐bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6‐bisphosphate are close to the sulfate ion near the P‐axis in the “apo” model.This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo‐enzyme showed far greater similarity to the ternary complex of dogfish M4 lactate dehydrogenase with incotinamide adenine dinucleotide and oxamate than to apo‐M4 dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose, 1,6‐bisphosphate were also analyzed.

Receptor-mediated endocytosis of lactate dehydrogenase M4 by liver macrophages: a mechanism for elimination of enzymes from plasma. Evidence for competition by creatine kinase MM, adenylate kinase, malate, and alcohol dehydrogenase.

We have previously shown that the rapid clearance of intravenously injected lactate dehydrogenase M4 from plasma is mainly due to endocytosis by macrophages in liver, spleen, and bone marrow. We have now studied endocytosis of lactate dehydrogenase M4 in detail, using freshly isolated rat liver macrophages (Kupffer cells) in vitro. 125I-lactate dehydrogenase M4 rapidly accumulated in the cells and was subsequently degraded to trichloroacetic acid-soluble material. Degradation was inhibited by leupeptin, an inhibitor of lysosomal proteases. Breakdown of the protein was also greatly diminished by treatment of the cells with chloroquine, a weak base which inhibits proteolysis by raising the pH in endosomes and lysosomes. High concentrations of chloroquine inhibited uptake. Lactate dehydrogenase M4 was not endocytosed by liver endothelial cells, although, under the same conditions, these cells were shown to accumulate horse radish peroxidase via a mannose-specific receptor. Uptake of lactate dehydrogenase M4 by Kupffer cells was strongly reduced after pretreatment of the cells with low concentrations of proteases. Endocytosis of lactate dehydrogenase M4 exhibited saturation kinetics (Km = 0.8 microM) and was competitively inhibited by mitochondrial and cytosolic malate dehydrogenase, alcohol dehydrogenase, adenylate kinase, and creatine kinase MM, enzymes which are rapidly cleared in vivo. Enzymes with long half-lives in plasma, namely lactate dehydrogenase H4, alanine aminotransferase, and cytosolic aspartate aminotransferase did not compete at concentrations up to 10 microM. Our results indicate that Kupffer cells contain a receptor that is involved in the clearance of lactate dehydrogenase M4 and a number of other tissue-derived enzymes from plasma. Uptake of lactate dehydrogenase M4 does not occur via a receptor that recognizes carbohydrate residues, for the enzyme is not a glycoprotein.

Reexamination of metabolic potential in the toadfish sonic muscle.

Activities of eight enzymes were measured in the sonic muscle of the gulf toadfish, Opsanus beta, to determine the metabolic poise of this unique tissue and to evaluate potential sex related differences in metabolism. In contrast to a prior study (Pennypacker et al., '85, J. Exp. Zool., 239: 259-264), we observed substantial activities of M4-lactate dehydrogenase, 333 to 482 units/g wet sonic muscle weight. This observation and the presence of high activities of other enzymes of glycolytic and anaerobic metabolism (pyruvate kinase and creatine phosphokinase) lead us to conclude that this tissue has high anaerobic capacity. Also in contrast to the observations of Pennypacker et al. ('85), we found that the activities of some enzymes indicative of aerobic metabolism are relatively low. For example, the activities of citrate synthase found in sonic muscle (1.5 to 2.7 units/g) are only slightly higher than values obtained for toadfish white skeletal muscle (1.2 units/g). The discrepancies between the results obtained by the two studies appear to be methodological ones. Lastly, significant differences in enzyme activities between males and females were observed for lactate dehydrogenase, malate dehydrogenase, and citrate synthase, and possible explanations for these differences are discussed.

Characterization of a lactate dehydrogenase inhibitor protein from rabbit skeletal muscle mitochondrial fraction

A lactate dehydrogenase inhibitor protein is isolated from rabbit skeletal muscle crude mitochondrial fraction. The molecular weight of the inhibitor is approximately 20,000 as determined by size exclusion HPLC. The inhibitor isoelectricpH is 5.3 as determined by agarose or polyacrylamide gel isoelectric focusing. The amino acid composition of the inhibitor is given. The presence of the inhibitor gives an acidic characteristic to the alkaline M4 lactate dehydrogenase isozyme and the lactate dehydrogenase-inhibitor complex is more stable than the enzyme alone.

Pinocytosis and degradation of exogenous proteins by cystinotic fibroblasts

Lysosomes of cystinotic human fibroblasts contain over 100-times the normal concentration of cystine. The high cystine concentration (probably in the millimolar range) might be expected to inhibit intralysosomal protein breakdown. A comparison of pinocytosis and degradation of five 125I-labelled proteins (bovine serum albumin, formaldehyde-denatured bovine serum albumin, bovine pancreatic ribonuclease A and porcine lactate dehydrogenase isoenzymes H4 and M4) by human fibroblasts has been made, using one cystinotic and two normal cell-lines. The proteins each entered fibroblasts by adsorptive pinocytosis and were then degraded within the lysosomes by enzymes susceptible to leupeptin, the thiol-proteinase inhibitor. Each protein was captured by the fibroblasts at a characteristic rate, which was not different in cystinotic cells. Normal and cystinotic fibroblasts did not differ in their proteolytic capacity, as measured in extracts of disrupted cells. In intact fibroblasts, four of the five proteins were rapidly and fully digested following pinocytosis, in both cystinotic and normal cells. However, with formaldehyde-denatured albumin, the most resistant to degradation of the proteins tested, or with some other proteins in the presence of leupeptin, when the proteolytic capacity of lysosomes is diminished, intralysosomal degradation of pinocytosed protein was incomplete. Moreover, under these conditions, cystinotic cells demonstrated a lower rate of protein digestion than normal cells. It is concluded that pinocytic capture, rather than intralysosomal proteolysis, is commonly the rate-limiting step in the overall process of uptake and degradation of proteins by fibroblasts, and that intralysosomal cystine inhibits digestion of pinocytosed protein only in circumstances when degradation becomes the rate-limiting step.

Several dehydrogenases and kinases compete for endocytosis from plasma by rat tissues.

Plasma contains many enzymes that are probably derived from damaged cells. These enzymes are cleared at characteristic rates. We showed previously that in rats the rapid clearance of alcohol dehydrogenase, lactate dehydrogenase M4 and the mitochondrial and cytosolic isoenzymes of malate dehydrogenase is largely due to endocytosis by macrophages in liver, spleen and bone marrow. We now demonstrate that uptake of each of the enzymes by these tissues is in general decreased by simultaneous injection of a high dose of one of the other dehydrogenases or a high dose of adenylate kinase or creatine kinase. A similar dose of colloidal albumin did not significantly decrease uptake of the four dehydrogenases. Nor was uptake of colloidal albumin, apo-peroxidase from horseradish or multilamellar liposomes influenced by a high dose of mitochondrial malate dehydrogenase. These results indicate that the four dehydrogenases and the two kinases are specifically endocytosed via the same receptor. We suggest that this receptor contains a group, possibly a nucleotide, with affinity for the nucleotide-binding sites of the enzymes.

Progress in the refinement of apo M4lactate dehydrogenase from dogfish (Squalus acanthias)

Progress in the refinement of apo M₄lactate dehydrogenase from dogfish (<i>Squalus acanthias</i>)

Plasma clearance and endocytosis of cytosolic malate dehydrogenase in the rat.

1. Pig heart cytosolic malate dehydrogenase was radiolabelled with O-(4-diazo-3,5-di-[125I]iodobenzoyl)sucrose and intravenously injected into rats. Enzyme activity and radioactivity were cleared from plasma identically, with first-order kinetics, with a half-life of about 30 min. 2. The tissue distribution of radioactivity was determined at 2 h after injection. All injected radioactivity was recovered from the tissues. A high percentage of the injected dose was found in liver (37%), spleen (6%) and bone including marrow (19%). 3. Radioactivity in liver and spleen increased up to 2 h after injection and subsequently declined, with a half-life of about 20 h. 4. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. 5. Liver cells were isolated 1 h after injection of labelled enzyme. We found that Kupffer cells, endothelial cells and parenchymal cells had endocytosed the enzyme at rates corresponding to 2725, 94 and 63 ml of plasma/day per g of cell protein respectively. 6. Radioautography indicated that in spleen and bone marrow the enzyme is mainly taken up by macrophages. 7. Internalization of the enzyme by liver, spleen and bone marrow was saturable. This indicates that the enzyme is taken up in these tissues by adsorptive endocytosis. 8. The present results closely resemble those obtained previously for the mitochondrial isoenzyme of malate dehydrogenase and for lactate dehydrogenase M4. Since those enzymes are positively charged at physiological pH, whereas cytosolic malate dehydrogenase is negative, net charge cannot be the major factor determining the rate of uptake of circulating enzymes by reticuloendothelial macrophages, as has been suggested in the literature [Wachsmuth & Klingmüller (1978) J. Reticuloendothel. Soc. 24, 227-241].

Loggerhead turtle lactate dehydrogenases. How general is the apparent adaptation to prolonged anaerobiosis displayed by the lactate dehydrogenase isoenzymes from turtles of the genus Pseudemys?

1. 1. The lactate dehydrogenase M4 and H4 isoenzymes of the loggerhead turtle Caretta caretta were purified by oxamate affinity chromatography. 2. 2. The kinetic properties of these lactate dehydrogenases resemble those of the homologous homopolymers of most other vertebrates. 3. 3. Unlike the lactate dehydrogenases from turtles of the genus Pseudemys, the Caretta caretta M4 and H4 isoenzymes do not display similar sensitivities to substrate inhibition by pyruvate.

A quantitative study of the biospecific desorption of rat liver (M4) lactate dehydrogenase from 10-carboxydecylamino-Sepharose. Determination of the number of ligand-binding sites blocked on adsorption

1. The theory of Nichol, Ogston, Winzor & Sawyer [(1974) Biochem. J. 143, 435-443] for quantitative affinity chromatography, when adapted for use with a non-specific column from which a multi-site protein can be specifically desorbed by its free ligand, permits determination of the concentration of adsorption sites on the column, their adsorptive affinity (as an association constant) and either the intrinsic (site) constant for ligand-binding to the protein or an 'occlusion coefficient' (defined as the number of ligand-binding sites blocked on adsorption), one of which must be known. 2. The theory has been applied to the NADH-specific desorption of rat liver M4 lactate dehydrogenase from 10-carboxydecylamino-Sepharose. It suggests that most of the enzyme molecules are adsorbed with at least two NADH-binding sites blocked, indicating an extensive adsorption interface in relation to the protein surface. Other chromatographic parameters were also determined for the system. 3. Among topics discussed are (a) factors affecting the experimentally determined value for the number of blocked sites, (b) the nature of the adsorption sites on the column and (c) the similarity of the analysis to that for determining Hill coefficients, and other possible applications.

Purification of M4 lactate dehydrogenase from rat liver by using biospecific desorption from 10-carboxydecylamino-Sepharose

Purification of M4 lactate dehydrogenase from rat liver by using biospecific desorption from 10-carboxydecylamino-Sepharose

Crystallization and preliminary crystallographic analysis at low resolution of the allosteric l-lactate dehydrogenase from Lactobacillus casei

The allosteric l-lactate dehydrogenase from Lactobacillus casei has been crystallized in its complex with the activators fructose-1,6-diphosphate and Co 2+. The enzyme crystallizes in space group C2 with six tetramers in the unit cell. At very low resolution, 00 l reflexions are absent for l ≠ 3 n. The orientation of the molecular axes has been determined using the rotation function. All tetramers in the unit cell exhibit excellent 222 symmetry, and the overall arrangement resembles the packing that would be expected in the higher symmetry space group P3 121. Comparison with the apo-enzyme structure of M4-lactate dehydrogenase from dogfish indicates high structural similarity between these enzymes and allowed us to identify the molecular axes of L. casei l-lactate dehydrogenase in terms of the “standard” molecular co-ordinate system P, Q, R. The similarity of both enzymes is good enough to allow the structure determination of L. casei l-lactate dehydrogenase by molecular replacement using the dogfish enzyme as a model. Sequencing results show that L. casei l-lactate dehydrogenase is lacking the N-terminal arm of vertebrate lactate dehydrogenases and electron density maps at 5 Å resolution indicate that ligands might possibly bind in the region of the missing arm. The active site loop is involved in intermolecular contacts and its structure might be different from both, apo- and ternary dogfish l-lactate dehydrogenase.