Additional lactate dehydrogenase (LDH) isoenzymes in normal testis and spermatozoa of adult man

Summary Adult human testicular tissue contains up to six previously undescribed lactate dehydrogenase (LDH) isoenzymes in addition to the five LDH isoenzymes normally found and the sixth found in spermatogenic cells and spermatozoa, LDH-X. Additional LDH isoenzymes were also found in spermatozoa but not in seminal fluid or in serum. After electrophoresis one additional LDH isoenzyme of testicular tissue was localized between LDH-1 and LDH-2, two between LDH-2 and LDH-3, two between LDH-3 and LDH-4, and two between LDH-4 and LDH-5. These localizations indicate that the additional LDH isoenzymes are tetramers combining the A and B subunits of the five normal LDH isoenzymes and the C subunit of LDH-X. The additional LDH isoenzymes may be important in the metabolism of spermatogenic germ cells and spermatozoa.     

Predation and competition within an assemblage of larval newts (Triturus)

The impact of crested newts (Triturus cristatus) on the smaller-bodied palmate and smooth newts (T helveticus and T vulgaris) was studied during the larval stages using a combination of field and laboratory experiments In pond enclosures T cristatus larvae had no effect on the two smaller species over the first four weeks of development By eight weeks, however. T cristatus had achieved a size advantage which enabled it to eliminate T helveticus and severely reduce T vulgaris by predation In laboratory trials under food-limited conditions, T helveticus and T vulgaris were slightly smaller when raised with T cristatus, suggesting that this predatory effect was complemented by interspecific competition during early development Predation of the smaller species started when T cristatus reached a threshold size of c 27 mm No reciprocal effects on T cristatus growth or survival were observed Although T cristatus may be a significant predator of congeneric species in natural ponds, other factors, such as differences in microhabitat selection, higher-order predator-prey interactions and the occasional desiccation of pond habitats may facilitate coexistence between the species     

Evidence for the lack of subunit exchange between aldolase tetramers in vivo.

The results of a double isotope experiment using 3H- and 14C-labeled leucine as precursors of protein synthesis demonstrated that the aldolase C to A subunit transition which is associated with chick skeletal muscle development involves the preferential synthesis of different aldolase isoenzymes. This developmental system was used to test for subunit exchange between aldolase tetramers in vivo. In a second double isotope experiment, it was found that the 14C:3H ratios of A and C subunits derived from the same heterotetramer were essentially identical, while the isotope ratios of the same subunit type derived from different isoenzymes were considerably different. Had subunit exchange between the isoenzymes occurred, A subunits of a given heterotetramer would have been expected to have higher isotope ratios than the corresponding C subunits. Therefore, these data suggest that subunit exchange between aldolase tetramers does not occur in vivo, at least not in skeletal muscle to an appreciable extent. The results of the present study suggest that all aldolase tetramers are constructed at the time of the initial assembly of newly synthesized subunits, that is, "new" tetramers would not be generated by subunit exchange between already constructed tetramers. In addition, the present work suggests that the degradation of all four subunits of an aldolase tetramer are coupled inasmuch as the subunits would not be reincorporated into other tetramers. Thus, in contrast to some other proteins, it appears that the subunits of the aldolase tetramer turn over coordinately.     

Estimation of correlation of lactate dehydrogenase subunits mole quota based on differences in substrate inhibition

Summary A kinetic method of estimating the mole quota ratios of the human lactate dehydrogenase (LDH) H and M subunits based on differences in substrate inhibition of LDH isoenzymes by lactate is proposed. Stability of kinetic constants for a prolonged period of time is demonstrated. The dependence of the activity ratios on the contribution of the mole quota of the M-subunit of LDH is studied under conditions of low and high substrate concentrations. The experimental and theoretical values show the following correlation: r = 0.998; p < 0.001. A comparison of the method proposed with the electrophoretic method of LDH subunit estimation is made, the values obtained being in good agreement. No effect of the components of human diploid cell homogenate and only an insignificant effect of the blood serum components on the kinetic constants of LDH isoenzymes are shown. The applicability of the method to the estimation of the quantitative content of both LDH subunits in natural samples is demonstrated. The informational value of the method is compared to that of other standard methods of LDH isoenzyme estimation.The need of the rapid and reliable method for determination the lactate dehydrogenase (LDH) activity of the H and M subunits has long been a matter of great importance, since the study of LDH isoenzymes is an indispensable part of clinical, genetical, cytological and herontological investigations.In 1960 PLAGEMANN et al. ¹ ,making use of different substrate inhibition of H₄ and M₄ isoenzymes LDH, developed a method for the estimation of the percent composition H and M subunits LDH within any given mixture of them. The method involves the assay of mixture of LDH isoenzymes in the presence of two different levels of pyruvate. The authors calculated the percent of each subunit in a mixture from the ratio of enzymatic activities at both high and a low concentration of pyruvate. Although this method was subsequently improved, both experimentally^2–S and theoretically⁶, its application was still impossible without first eliminating a great many problems. The problem of subunit interactions inside the enzyme molecule has not been settled. In addition, questions have not been raised about stability of the kinetic parameters', the reproducibility of the method, its applicability to the study of different objects and also the informational value of the experimental data.In our previous investigation^7,8, we have studied the kinetic properties of five purified isoenzymes of human lactate dehydrogenase and demonstrated the catalytic independence of the active sites of the LDH tetrameric molecules with respect to substrate inhibition.In the present report an attempt has been made to develop a kinetic method for the assay of M-polypeptide chains mole quotum of lactate dehydrogenase in natural specimens.     

树鼩血清乳酸脱氢酶同工酶的研究

采用0.5％琼脂糖凝胶电泳技术对70例云南树鼩（Tupaia belangeri chinensis）正常血清乳酸脱氢酶（LDH）同工酶进行电泳分析。结果表明,树鼩血清乳酸脱氢酶呈现5种不同分子形式的同工酶表型。其LDH₄至LDH₁同工酶随电泳迁移率的增大依次趋于阳极端,LDH₅同工酶移向阴极端。采用分光光度定量法分别测得雌雄个体血清LDH₁-LDH₅5种不同分子形式同工酶的相对百分含量分别为17.9,14.5,20.6,19.7,27.4,和19.6,15.2,18.3,17.7,29.5,雌雄个体H/M亚基比率分别为0.78和0.79。     

Lactate and malate dehydrogenases in the muscles and male genital tract of the rabbit.

Average lactate dehydrogenase (LDH) isoenzyme patterns the content of H subunits, total LDH activity, total malate dehydrogenase (MDH) activity and the m- MDH/s-MDH ratio were determined in twelve muscles and the male genital tract of the rabbit. LDH(1) was the predominant form in the heart, soleus and masseter muscles, LDH(3) in the lingual muscles and LDH(5) in the other muscles analysed. In the muscles, an increase in the percentual proportion of M subunits was accompanied, by a proportional increase in total LDH activity and a decrease in total MDH activity, especially m-MDH. LDH isoenzyme patterns and LDH and MDH activities are useful for obtaining some idea about the proportion of individual muscle fibres. Activity accounted for by H subunits was roughly the same in all the muscles analysed, indicating that the synthesis of H subunits is independent of the type of muscle fibre and of the oxygen supply of the muscular tissue, and also that isoenzymes composed chiefly of H subunits are not localized preferentially in the mitochondria. Similar relationships between LDH isoenzymes and LDH and MDH activities were found in the testicular and epididymal tissues. The tests and the head of the epididymis mainly contain LDH isoenzymes composed of H subunits. The total LDH activity in these tissues is relatively low and their MDH activity is relatively high compared with the body and tail of the epididymis. The proportion of H subunits in the ampulla, the seminal vesicles, the coagulating glands and the prostate is also high. Cowper's glands have a high LDH(5) and LDH(4) concentration. One of two LDHx isoenzymes were found in the testes and spermatozoa.     

The effect of pH on feeding behaviour in newt larvae (Triturus: Amphibia)

The feeding responses of three species of newt larvae were compared under circumneutral and sublethal acid conditions. Under acid conditions (pH 4.5) feeding behaviour was suppressed in palmate newts, Triturus helveticus, and smooth newts. T. vulgaris , but not in crested newts, T. cristatus. At low pH, approach and orientation towards food occurred in T. helveticus and T. vulgaris , but snapping was inhibited; T. cristatus snapped and consumed food immediately it was offered under the same conditions. These differences are not consistent with the apparent greater tolerance of T helveticus for acidified ponds. The observations suggest that the chemosensory system of T. helveticus and T. vulgaris may be impaired at low pH.     

大豆黄酮对衰老小鼠脑组织SOD、LDH同工酶的影响

利用SOD和LDH同工酶电泳分析,研究大豆黄酮对衰老小鼠的抗氧化作用。结果显示大豆黄酮没有改变SOD和LDH同工酶谱的特征,但对因衰老引起的小鼠脑组织LDH和SOD同工酶活性、各组分的相对活性和比活力的变化有不同程度的改善作用,即LDH同工酶中LDH-2、LDH-3的活性明显下降,LDH-1的活性下降最为明显,而LDH-4的活性有所下降,但不显著,LDH-5的活性几乎没有变化,SOD同工酶的SOD-1和SOD-2的活性有不同程度的升高。这表明大豆黄酮是通过抑制LDH同工酶H亚基的合成来降低LDH的活性,而对M亚基的合成没有影响,并且能够促进SOD同工酶SOD-1和SOD-2的合成,不影响其遗传稳定性。     

Differential effects of pH and temperature on embryonic development in the British newts (Triturus)

Changes in the water quality and temperature relationships of ponds may affect the structure of amphibian assemblages. The survival, time to hatching, hatching size and hatching stage of newt embryos were studied in the three British species ( Triturus cristatus, T. helveticus and T. vulgaris ), at two temperatures and two pHs. All T. cristatus embryos failed to hatch at pH 4.5, whereas over 80% of T. helveticus and T. vulgaris embryos hatched successfully at the same pH. At pH 7.5, T. cristatus survival was the same as the other two species, after the 50% mortality due to the homomorphism of chromosome 1 was taken into account. Temperature had no effect on survival of embryos. Time to hatching was two to four times longer at 12°C than at 17°C. Low pH shortened development time in T. vulgaris but not in T. helveticus . Low pH, but not temperature, affected size at hatching, with T. helveticus and T. vulgaris embryos emerging at a smaller size and earlier stage of development under acidic conditions. This reduction of size at low pH affected T. vulgaris more than T. helveticus . We predict that T. cristatus embryos will be the most vulnerable of the three species to acidification in nature. Warm ponds will result in rapid embryonic development, but T. helveticus and T. vulgaris larvae hatching in acid ponds will do so at a smaller size and earlier stage of development. The pattern of vulnerability to acidification within amphibian assemblages may change during ontogeny.     

Epigenetic formation of lactate dehydrogenase isozymes in the house mouse, Mus musculus.

The origin of mouse lactate dehydrogenase (LDH) sub-bands was investigated by using our miniaturized polyacrylamide gel electrophoretic apparatus. Mouse LDH isozymes are generated by combinations of three types of A subunit, the primary type and two epigenetically modified forms. These are designated A1, A2, and A3 in the order of their electrophoretic mobilities towards the anode. The A1 subunit arises from the covalent binding of molecules of glutathione through disulfide bonds to the original subunit, A3. The A2 subunit arises from the covalent binding of molecules of cysteine through disulfide bonds to the A3 subunit. All isozymes can be explained as tetramers composed of the three kinds of A subunit (A1, A2, or A3) in combination with B subunits to yield a total of 35 isozymes. The kinetic properties of these sub-bands were also examined. There was no difference between A24 and A34 in the Km for pyruvate and for lactate. Thermostability at 56 degrees C was greater for A34 than for A24. The activities of tetramers at the electrophoretic position of A3B1 and A4 in extracts containing all five isozymes were increased by treatment of the extracts with high concentrations of reduced glutathione or cysteine with the concomitant disappearance or decrease in activity of tetramers at the position of B4 and A3B1. These results suggest that, in the presence of reduced glutathione or cysteine, LDH isozymes containing the B subunit are first dissociated and then the A subunits are preferentaially recombined.     

Stability of quaternary structure and mode of dissociation of fructosediphosphate aldolase isoenzymes

Using a highly sensitive "subunit exchange" assay, we have studied the relative strengths of interactions between different subunit types (A and C) of fructosediphosphate aldolase and have determined the mode of dissociation of aldolase tetramers in vitro. Interactions between C subunits within C4 tetramers were found to be considerably more resistant to disruption than were interactions between A subunits in A4 tetramers with regard to increasing concentrations of H+, OH-, or urea. Slight dissociation of A4 was also observed in 1.2 M magnesium chloride. These observations suggest that the quaternary structure of aldolase C4 is inherently more stable than that of aldolase A4. Also, the symmetrical heterotetramer A2C2 was found to be more resistant to urea-mediated dissociation than was the aldolase A4 homotetramer; this observation suggests that, even when in heteromeric combination, C subunits have a stabilizing influence on the quaternary structure of aldolase tetramers. In no case did we find evidence for a stable dimeric intermediate in the dissociation of aldolase tetramers to monomers. These observations are considered in terms of the tetrahedral arrangement of subunits in the aldolase tetramer. The general applicability of the subunit exchange assay described here for studying the subunit structure and mode of dissociation of oligomeric enzymes is discussed.     

Some molecular properties of rat-liver lysosomal beta-glucuronidase isoenzymes.

The isoenzymes of rat-liver lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase (EC 3.2.1.31)) were inactivated at different rates at 0 degrees C in 3M guanidinium chloride solutions adjusted to pH 5.0 In 4 M urea buffered by 0.01 M glycylglycine, pH 7.0 isoenzymes I, III, and V were reversibly inhibited 80%. Sodium dodecyl sulfate (SDS), 0.1% in 0.01 M phosphate buffer, pH 7.0 irreversibly inhibited at 37 degrees C all five isoenzymes. Sedimentation analysis showed that loss of catalytic activity in these denaturing media is accompanied by dissociation into slower sedimenting subunits. SDS gel electrophoresis revealed that the isoenzymes are apparently tetramers made up of different proportions of subunits alpha, beta, and gamma having apparent molecular weights of 62,900, 60,200, and 58,700, respectively. The three subunits appear to be glycoproteins.     

Properties of the interspecies hybrids between haptoglobin alpha and beta subunits

1. Subunits alpha isolated from human haptoglobin were recombined with beta subunits of equine haptoglobin, and vice versa. Both hybrid proteins were separated on electrophoresis in polyacrylamide gel into four bands with mobilities corresponding to tetramers 2alpha.2beta, trimers 2alpha.beta, and dimers alpha.beta, in addition to free subunits beta. 2. The binding ability of haemoglobin and the antigenic specificity of tetramers depended on the origin of beta subunit. 3. Reduction of native and hybrid proteins with 2-mercaptoethanol led to gradual formation of alpha.beta, alpha, and beta; the components 2alpha.beta and 2alpha appeared in trace amounts.     

Lactate dehydrogenase binding to the mitochondrial fraction and to a mitochondrial inhibitor as a function of the isoenzymatic composition

Purified rabbit skeletal muscle LDH M4 isoenzyme, but not H4 isoenzyme, was observed to bind to either the crude mitochondrial fraction or a mitochondrial inhibitor. Several sources of LDH isoenzymes in which M-type subunits with an alkaline pI are predominant bind to this crude mitochondrial fraction and are inhibited by the mitochondrial inhibitor. Binding and inhibition have also been observed with H-type isoenzymes with a pI near 7. The binding and the inhibition processes did not occur with H-type isoenzymes with an acid pI or with M-type isoenzymes with pI near 6. The binding capacity of LDH to the mitochondrial fraction and to the mitochondrial inhibitor is very similar and depends on the net protein charge and not on whether the subunits are H- or M-type.     

Lactate dehydrogenase: inhibition of subunit A by the sulfhydryl reagent AgNO3.

Lactate dehydrogenase isoenzymes including the subunit A--LDH 5 (A4), LDH 4 (A3B), LDH 3 (A2B2), and LDH 2 (AB3)--are inhibitable by the sulfhydryl reagent AgNO3, while the isoenzyme LDH 1 (B4) is not.     

Isoenzymes of phosphofructokinase in the rat. Demonstration of the three non-identical subunits by biochemical, immunochemical and kinetic studies.

In man and the rabbit, 6-phosphofructokinase (PFK, EC 2.7.1.11) exists in tetrameric isoenzymic forms composed of muscle (M or A), liver (L or B) and platelet or brain (P or C) subunits, which are under separate genetic control. In contrast, the genetic control of the rat PFK has not yet been conclusively established; it is unclear whether the P-type or C-type subunit exists in this species. To resolve this question, we investigated the enzyme from the skeletal muscle, liver and brain of rats of Wag/Rij strain. Our studies demonstrate that the rat PFK is also under the control of three structural loci and that the homotetramers M4, P4 and L4 exhibit unique chromatographic, immunological and kinetic-regulatory properties. Skeletal-muscle and brain PFKs consist of isolated M4 and P4 homotetramers respectively. Although liver PFK consists predominantly of L4 homotetramer, it also contains small amounts of PL3 and P2L2 species. All three PFKs exhibit allosteric properties: co-operativity with fructose 6-phosphate and inhibition by ATP decrease in the order P4 greater than L4 greater than M4. P4 and M4 tetramers are the most sensitive to citrate inhibition, whereas L4 tetramer is the least sensitive. More importantly, P4 and L4 isoenzymes are the most sensitive to activation by fructose 2,6-bisphosphate, whereas M4 isoenzyme is the least sensitive. These results indicate that the brain PFK in this strain of rat is a unique tetramer, P4, which also exhibits allosteric kinetics, as do the well-studied M4 and L4 isoenzymes. The reported differences in the number and nature of isoenzymes present in the rat brain and liver most probably reflect the differences in the strains studied by previous investigators. Since the nature of the rat PFK isoenzymes and nomenclatures reported by previous investigators have been now reconciled, it is proposed that, for the sake of uniformity, only well-established nomenclatures used for the rabbit or human PFK isoenzymes be used for the rat isoenzymes.     

The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts.

1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.     

On intratetrametric catalytic independency of active sites of lactate dehydrogenase isoenzymes]

Interrelations of active sites of tetrameric molecules of human lactatedehydrogenase (LDH), known as intratetrameric catalytic independency of subunits, are studied. Estimation of catalytic activity of subunits, which compose hybrid LDH isoenzymes, is carried out. Ratios of molecular activity of subunits are calculated and a conclution is drawn on the catalytic independency of LDH isoenzymes active sites with respect to substrate inhibition by L-lactate. Possible mechanisms of substrate inhibition of LDH isoenzymes and their inactivation with urea in the view of different interrelations of active sites of these isoenzymes under conditions studied are discussed.     

Active site ligand stabilization of quaternary structures of glutamine synthetase from Escherichia coli

Auto-inactivated EScherichia coli glutamine synthetase contains 1 eq each of L-methionine-S-sulfoximine phosphate and ADP and 2 eq of Mn2+ tightly bound to the active site of each subunit of the dodecameric enzyme (Maurizi, M. R., and Ginsburg, A. (1982) J. Biol. Chem. 257, 4271-4278). Complete dissociation and unfolding in 6 M guanidine HCl at pH 7.2 and 37 degrees C requires greater than 4 h for the auto-inactivated enzyme complex (less than 1 min for uncomplexed enzyme). Release of ligands and dissociation and unfolding of the protein occur in parallel but follow non-first order kinetics, suggesting stable intermediates and multiple pathways for the dissociation reactions. Treatment of Partially inactivated glutamine synthetase (2-6 autoinactivated subunits/dodecamer) with EDTA and dithiobisnitrobenzoic acid at pH 8 modifies approximately 2 of the 4 sulfhydryl groups of unliganded subunits and causes dissociation of the enzyme to stable oligomeric intermediates with 4, 6, 8, and 10 subunits, containing equal numbers of uncomplexed subunits and autoinactivated subunits. With greater than 70% inactivated enzyme, no dissociation occurs under these conditions. Electron micrographs of oligomers, presented in the appendix (Haschemeyer, R. H., Wall, J. S., Hainfeld, J., and Maurizi, M. R., (1982) J. Biol. Chem. 257, 7252-7253) suggest that dissociation of partially liganded dodecamers occurs by cleavage of intra-ring subunit contacts across both hexagonal rings and that these intra-ring subunit contacts across both hexagonal rings and that these intra-ring subunit interactions are stabilized by active site ligand binding. Isolated tetramers (Mr = 200,000; s20,w = 9.5 S) retain sufficient native structure to express significant enzymatic activity; tetramers reassociate to dodecamers and show a 5-fold increase in activity upon removal of the thionitrobenzoate groups with 2-mercaptoethanol. Thus, the tight binding of ligands to the subunit active site strengthens both intra- and inter-subunit bonding domains in dodecameric glutamine synthetase.     

岳鲤及其双亲(荷包红鲤♀、湘江野鲤♂)LDH同工酶的研究

用聚丙烯酰胺凝胶电泳,结合CS910双波长扫描分析了岳鲤及其双亲(荷包红鲤♀、湘江野鲤♂)6种组织的乳酸脱氢酶同工酶(Lactate Dehydrogenase,LDH)。结果表明:岳鲤及其双亲的LDH同工酶均存在组织特异性;亲本之间LDH同工酶的差异主要表现在肝脏;亲本各组织的大多数LDH同工酶在F_1代得到表现;亲本及F_1代的LDH同工酶可能由基因位点AA′BB′共同控制。本文还讨论了LDH同工酶分析和杂交结果预测的关系。