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PubMed1994

Involvement of a cystatin-alpha-sensitive cysteine proteinase in the degradation of native L-lactate dehydrogenase and serum albumin by rat liver or kidney lysosomes.

A lysosomal cysteine proteinase plays a critical role in the lysosomal degradation of native L-lactate dehydrogenase. The effects of a recombinant cystatin alpha and other cysteine proteinase inhibitors on the degradation of unlabeled native L-lactate dehydrogenase by total lysosomal enzymes were examined in vitro. L-Lactate dehydrogenase was inactivated, its 35-kDa subunit disappeared and the final amino acid degradation products were produced during an incubation with disrupted lysosomes in vitro. These processes were all markedly suppressed by a small amount of cystatin alpha without inhibiting the activities of the known lysosomal cysteine proteinases, cathepsins B, H, L, and J. These results suggest that a cysteine proteinases, which is highly sensitive to cystatin alpha and distinct from other known lysosomal cysteine proteinases, is involved in the lysosomal degradation of native L-lactate dehydrogenase. An L-lactate-dehydrogenase-inactivating enzyme in the extracts of lysosomes was partially purified. It was separated from cathepsin J using a Sephacryl S-200 gel-filtration column and was further separated from cathepsin H by DEAE-Sephadex A-50 anion-exchange column chromatography. The inactivation and degradation of L-lactate dehydrogenase by this partially purified enzyme were all markedly suppressed by a low level of cystatin alpha without inhibiting the activities of both cathepsins B and L. The degradation of rat serum albumin by the partially purified enzyme was also inhibited by the same concentration of cystatin alpha. It is concluded that a cystatin-alpha-sensitive cysteine proteinase, other than cathepsins B, H, L and J, is present in lysosomes and functions in the lysosomal degradation of at least native L-lactate dehydrogenase and serum albumin.

Animals↗

PubMed2000

Lactate dehydrogenase A-dependent surface expression of immature thymocyte antigen-1: an implication for a novel trafficking function of lactate dehydrogenase-A during T cell development.

A possible involvement of lactate dehydrogenase A (LDHA) in the translocation of a thymic differentiation antigen, immature thymocyte antigen-1 (IMT-1), from cytoplasm to cell surface membrane during thymocyte differentiation is described. Transfection of cDNA for LDHA, but not LDHB, into EL4 cells, which expressed IMT-1 in the cytoplasm but not on the cell surface, induced the expression of IMT-1 on the cell surface. This translocation process seemed to be dependent on the translation of LDHA cDNA in EL4 cells, as well as the native structure of LDHA composing of coenzyme binding domain, catalysis domain, and subunit contact domain. Immunoprecipitation analysis revealed that LDHA could be co-precipitated with IMT-1 from the cell surface of EL4 cells that had been transfected with LDHA cDNA, suggesting that some of LDHA is associated with cell surface IMT-1 on the transfectants. Flow cytometry analysis of thymocyte subpopulations showed that some thymocytes at the CD4(-)CD8(-) double negative stage express both IMT-1 and LDHA on their surface. These data indicate that LDHA, in addition to its function in the metabolism in the glycolytic pathway, may have a novel function in the expression of a cell surface antigen during T cell development.

Animals↗

PubMed1999

Overexpression of monocarboxylate transporter and lactate dehydrogenase alters insulin secretory responses to pyruvate and lactate in beta cells.

Previous investigations revealed low activities of lactate dehydrogenase (LDH) and plasma membrane monocarboxylate transporters (MCT) in the pancreatic beta cell. In this study the significance of these characteristics was explored by overexpressing type A LDH (LDH-A) and/or type 1 MCT (MCT-1) in the clonal INS-1 beta cells and isolated rat islets. Inducible overexpression of LDH-A resulted in an 87-fold increase in LDH activity in INS-1 cells. Adenovirus-mediated overexpression of MCT-1 increased lactate transport activity 3.7-fold in INS-1 cells. Although overexpression of LDH-A, and/or MCT-1 did not affect glucose-stimulated insulin secretion, LDH-A overexpression resulted in stimulation of insulin secretion even at a low lactate concentration with a concomitant increase in its oxidation in INS-1 cells regardless of MCT-1 co-overexpression. Adenovirus-mediated overexpression of MCT-1 caused an increase in pyruvate oxidation and conferred pyruvate-stimulated insulin release to isolated rat islets. Although lactate did not stimulate insulin secretion from control or MCT-1-overexpressing islets, co-overexpression of LDH-A and MCT-1 evoked lactate-stimulated insulin secretion with a concomitant increase in lactate oxidation in rat islets. These results suggest that low expression of MCT and LDH is requisite to the specificity of glucose in insulin secretion, protecting the organism from undesired hypoglycemic actions of pyruvate and lactate during exercise and other catabolic states.

Adenoviridae↗

PubMed1998

About the pKa of the active-site histidine in flavocytochrome b2 (yeast L-lactate dehydrogenase).

Flavocytochrome b2 or L-lactate dehydrogenase from yeasts catalyzes the oxidation of L-lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner. When incubated in the presence of both L-lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved. During this reaction, the substrate alpha-hydrogen is transferred not only to the solvent but also in part to the keto acid, which acts as reverse substrate. Thus, when bound to the reduced enzyme, this hydrogen is sticky. In the context of a carbanion mechanism, it resides on Nepsilon of His373, the active site base. We have shown before that a correlation between the amount of intermolecular hydrogen transfer from [2-3H] lactate and the keto acid reverse substrate concentration enables the determination of the first-order rate constant, kHe, for exchange of the substrate-derived protein-bound hydrogen with bulk solvent (Urban P, Lederer F, 1985, J Biol Chem 260:11115-11122). In this work, we show that the exchange with the solvent appears to be independent of the phosphate buffer concentration in the range from 40 to 500 mM. It is thus probable that exchange occurs directly with water molecules. The second-order rate constant for exchange is then 0.16 (+/-0.03) M(-1) s(-1). Using the Eigen equation, this figure yields a pKa of 9.1+/-0.1 for His373 in the reduced enzyme, compared to a probable value of 6.0 or less in the oxidized enzyme (Suzuki H, Ogura YC, 1970, J Biochem 67:291-295). The mechanistic significance of these results is discussed.

Binding Sites↗

PubMed1987

Lactate dehydrogenase activity in the mitochondrial fraction of chicken liver: enzyme binding and kinetic behavior of soluble and bound enzyme.

Chicken liver crude mitochondrial fraction showed lactate dehydrogenase activity (6.5% of cytoplasmic enzyme). Most of the mitochondrial lactate dehydrogenase was solubilized by sonication of the mitochondrial fraction in 0.15 M NaCl, pH 6. Total extracted lactate deshydrogenase activity was 3-fold higher than the initial pellet activity. Different isoenzymatic compositions were observed for cytosoluble and mitochondrial extracted lactate dehydrogenase. The pI, values of the 5 lactate dehydrogenase isoenzymes were found to be independent of their origin. The cytosoluble lactate dehydrogenase and the separated H4,H3M and H2M2 isoenzymes were able to bind to the chicken liver mitochondrial fraction in 5 mM sodium phosphate buffered medium, and could be solubilized afterwards with 0.15 M NaCl, pH 6. The enzyme bound to the mitochondrial fraction was less active than the soluble one. Particle saturation by the bound enzyme occurred with all mitochondrial fractions assayed. According to the Langmuir isotherm, the non-sonicated mitochondrial fractions contain a single type of binding sites for lactate dehydrogenase; in contrast, the sonicated mitochondrial fraction should contain different binding sites. Chicken liver crude or sonicated active mitochondrial fractions showed a hyperbolic behavior with respect to NADH and a non-hyperbolic one with respect to pyruvate. This mechanism is different from the bi-bi compulsory order mechanism of the soluble enzyme. With hydroxypyruvate as the substrate, the active mitochondrial fraction fit a sequential mechanism but lost the rapid-equilibrium characteristics of the soluble enzyme.

Animals↗

PubMed1969

Factors affecting the lactate dehydrogenase isoenzyme pattern of cultured kidney-cortex cells.

1. The lactate dehydrogenase isoenzyme pattern of cultured calf kidney-cortex cells was correlated to growth phase, changes in oxygen supply, mean generation time and changes in nutritional supply. 2. During culture of free cells and intact explants the lactate dehydrogenase isoenzyme pattern changed towards a dominance of isoenzymes containing the M subunit. 3. Of the shift in monomer proportion, 58% occurred during the lag phase and 42% during the initial part of the exponential growth phase. During the stationary phase the shift in monomer proportion reversed slightly. It was possible to relate the observed shift in monomer proportion to the glycolytic rate. 4. Factors that depressed glycolysis decreased the shift in monomer proportion. Oxygen was found to limit the decrease in the H subunit/M subunit ratio caused by anaerobic culture in vitro. 5. The results obtained support the view that the altered lactate dehydrogenase isoenzyme pattern of urine in renal ischaemia may be explained by anaerobic changes in the lactate dehydrogenase isoenzyme pattern of cortical tubule cells.

Animals↗

PubMed1982

[Lactate dehydrogenase activity in the presence of certain structural analogs of the nicotinamide fragment of NAD].

The activity of lactate dehydrogenase from the cattle myocardium extract as well as of this enzyme crystalline form was studied in the presence of structural analogues of the nicotinamide fragment NAD-amide, diethyl amide and N-oxymethyl amide of nicotinic acid. It is shown that the rate of the reaction catalyzed with lactate dehydrogenase under the effect of amide and N-oxymethyl amide of nicotinic acid decreases to a different extent depending on their concentration. Nicotinic diethyl amide practically has no effect on the activity of lactate dehydrogenase. Ki for crystalline lactate dehydrogenase and myocardium lactate dehydrogenase in the presence of nicotinic amide is 0.23.10-3 and 1.13.10-3 M. respectively, in the presence of nicotinic N-oxymethyl amide-0.30.10-3 and 0.10.10-3 M.

Animals↗

PubMed1996

Levels of lactate dehydrogenase in osteoarthritic and failed total knee joints.

Synoval fluid lactate dehydrogenase may be used as a marker of joint inflammation. Macrophage phagocytosis may result in cell llysis and release of intracellular lactate dehydrogenase. This study demonstrates that synovial lactate dehydrogenase levels are markedly increased in failed total knee arthroplasty as compared to end-state osteoarthritic knee joints.

Humans↗

PubMed1977

Ontogenic characteristics of lactate dehydrogenase turnover in the mouse.

1. In order to investigate the ontogenic and turnover characteristics of lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) in the foetal mouse, activities of this enzyme and isoenzyme distributions have been measured during development, and the time sequence of incorporation of radioactive amino acids have been determined during the last weeks of gestation. 2. In the early foetal stages, the enzyme was present with low specific activity and in the form of lactate dehydrogenase-5 in all regions, but a marked increase in specific activity became evident about the seventeenth day of gestation, and was accompanied by the formation of tissue specific patterns of isoenzyme distribution. 3. Measurement of turnover parameters was undertaken by both double-label ([3H] and [14C]leucine) and pulse-chase ([3H]leucine) techniques. Both procedures provided indications that appreciable degradation may occur during embryogenesis, with general proteolysis proceeding at a faster rate than with lactate dehydrogenase, and with higher rates of degradation occurring in foetuses which were smaller than average. 4. This data has been discussed in relation to the regional variation in these characteristics and the significance to studies of growth and differentiation.

Animals↗

PubMed2005

Protein S100B release from rat brain slices during and after ischemia: comparison with lactate dehydrogenase leakage.

One hour of ischemia significantly increased protein S100B release from rat brain slices without altering lactate dehydrogenase leakage. Reoxygenation of the ischemic slices, however, increased the levels of these biochemical markers in the medium. Although removal of extracellular Ca(+2) ions from the medium did not alter the basal lactate dehydrogenase leakage from cortical slices, an excessive increase in basal protein S100B release was seen under this condition. Ischemia and/or reoxygenation induced enhancements in these markers were attenuated by removal of Ca(+2) ions from the medium. Ischemia significantly increased glutamate release, but neither ischemia nor reoxygenation induced rises in protein S100B and lactate dehydrogenase levels were altered by glutamate receptor antagonists. Rising the glutamate levels in the medium by each ouabain or exogenous glutamate, moreover, failed in exerting an ischemia like effect on protein S100B and LDH outputs. In contrast, exogenous glutamate added into the medium protected the slices against reoxygenation induced increments in protein S100B and lactate dehydrogenase levels. These results indicate that protein S100B has a greater sensitivity against ischemia than lactate dehydrogenase in in vitro brain slice preparations. Since neither exogenous glutamate nor enhancements of the extracellular glutamate levels by ouabain had an ischemia like effect, and since glutamate receptor antagonists were also unsuccessful, it seems unlikely that ischemia-induced increase in glutamate release is directly involved in protein S100B release or lactate dehydrogenase leakage determined in the present study.

Animals↗

PubMed1983

Linkage and polymorphism of a gene controlling lactate dehydrogenase in the rat.

The amount of lactate dehydrogenase subunit A in rat serum is under the control of the lactate dehydrogenase regulatory gene Ldr-1, and it is determined by two alleles, Ldr-1a (high) and Ldr-1b (low). Sex and LDH-A level segregate independently, so the Ldr-1 gene is autosomal. The genes for albino, red eye, hemoglobin, and Ldr-1 are linked (linkage group I), and their order is Ldr-1-Hbb-r-c. Our data put Ldr-1 45 cM from Hbb, and Hbb is 7 cM from c. Data in the literature separate r and c by 0.3 cM.

Alleles↗

PubMed1971

Influence of carboxylic acids on the stereospecific nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases of Leuconostoc mesenteroides.

Leuconostoc mesenteroides increased its lactic acid production from glucose threefold when malic acid was added to the culture. This increase resulted also in a reduction of the ratio of d-lactic acid to l-lactic acid (31.5 to 1.23). Addition of malic acid increased 6.5-fold the specific activity of nicotinamide adenine dinucleotide (NAD)-linked l-lactate dehydrogenase and increased 3.2-fold that of NAD-linked d-lactate dehydrogenase. The Michaelis constant (K(m)) for NAD of the NAD-linked l-lactate dehydrogenase increased with the addition of malate, but no change was observed in the K(m) values for the respective d-enzyme. The effect of carboxylic acids on the NAD-linked l-lactate dehydrogenase activities was tested by using partially purified enzyme preparations from cells grown with glucose alone and from cells grown with glucose plus malate. Malate stimulated the l-enzyme and inhibited the d-lactate dehydrogenase. The NAD-linked l-lactate dehydrogenase exhibited the same activity bands on polyacrylamide gel electrophoresis whether the cell-free preparation originated from cells grown on glucose plus malate or on glucose as the sole carbon source. The NAD-linked d-lactate dehydrogenase, however, exhibited a different pattern of electrophoretic mobility, depending upon the source of origin of the cell-free preparation. The results suggest that malate has a stimulatory effect on the synthesis of both enzymes and may result in rearrangement of the protein structure of the d-lactate dehydrogenase. This rearrangement apparently makes the d-enzyme more susceptible to inhibition of catalytic activity. The l-lactate dehydrogenase, however, is stimulated not only in its synthesis but also in its activity. It is proposed that these effects are responsible for the regulation of lactic acid production.

Acids↗

PubMed1995

Stabilizing effect of sucrose against irreversible denaturation of rabbit muscle lactate dehydrogenase.

Measurements of the kinetics of activity loss by rabbit muscle lactate dehydrogenase in acetate-chloride buffer, pH 5.0, I 0.20, have shown that the enzyme exhibits greater stability against irreversible inactivation when the buffer is supplemented with sucrose (0.1 M-0.5 M). On the basis of sedimentation equilibrium distributions obtained for enzyme in the absence and presence of sucrose (0.5 M), the lactate dehydrogenase is essentially dimeric in both environments. The observed stabilization of enzyme activity has therefore been considered in terms of the space-filling effects of sucrose on an isomerization equilibrium between native and unfolded forms of dimeric lactate dehydrogenase, which precedes irreversible inactivation of the unfolded isomer. Interpretation of the kinetic results on that basis has led to the conclusion that the initial stage of enzyme unfolding entails a minor change in volume and/or asymmetry of the lactate dehydrogenase that gives rise to a 4% increase in the second virial coefficient describing excluded volume interactions between dimeric enzyme and sucrose.

Animals↗

PubMed1994

Ultrathin-layer microelectrophoretic determination of lactate dehydrogenase isoenzymes in corneal and conjunctival epithelium of the cow.

In order to evaluate the impact of tissue oxygenation on the distribution pattern of lactate dehydrogenase isoenzymes, activities of the isoenzymes were measured in microdissected samples of bovine tissue. A highly sensitive ultrathin-layer electrophoretic technique was used to determine the distribution pattern of lactate dehydrogenase isoenzymes in basal, intermediate and superficial layers of the epithelium of central and peripheral cornea and in the epithelium of the bulbar conjunctiva. Measurements revealed almost homogeneous intraepithelial distribution patterns of lactate dehydrogenase isoenzymes in both tissues. In the cornea the lactate dehydrogenase isoenzymes 4 and 5, which are regarded to be specialized for anaerobic glucose metabolism, were found to predominate. In the well-oxygenated conjunctival epithelium most of the activity could be ascribed to the lactate dehydrogenase isoenzyme 3. In contrast to the isoenzymatic activities, total activity of lactate dehydrogenase was inhomogeneously distributed; maximum activities were found in the basal layer of corneal epithelium and in the intermediate layer of conjunctival epithelium. The results indicate that oxygen supply is relevant rather for the intraepithelial distribution of total enzyme activity than for the expression of lactate dehydrogenase isoenzymes.

Animals↗

PubMed1988

Lactate dehydrogenase isoenzymes are present in matrix vesicles.

Matrix vesicles were isolated from epiphyseal growth plates of young rabbits. Lactate dehydrogenase activity was detected in the isolated matrix vesicles only in the presence of detergents, suggesting that NADH, the cofactor for the assay, does not penetrate the membrane of matrix vesicles. In contrast, the activity of alkaline phosphatase, a marker enzyme of the outer surface of matrix vesicles, was detected in the matrix vesicles using p-nitrophenyl phosphate as the substrate both in the presence and absence of detergents. Lactate dehydrogenase activity was detected only in the cytosol of chondrocytes of the epiphyseal growth plates but not in other subcellular fractions, showing that lactate dehydrogenase is not from the plasma membrane and membranes of intracellular organelles of chondrocytes. The isolated matrix vesicles contained all five lactate dehydrogenase isoenzymes but did not possess other cytosolic enzymes. These results show that lactate dehydrogenase is located in the matrix vesicles and suggest the presence of a mechanism for the specific uptake of cytosolic lactate dehydrogenase and the possibility of enzymatic quantification of the matrix vesicles at various calcification sites.

Animals↗

PubMed1992

Design of a specific phenyllactate dehydrogenase by peptide loop exchange on the Bacillus stearothermophilus lactate dehydrogenase framework.

Restriction sites were introduced into the gene for Bacillus stearothermophilus lactate dehydrogenase which enabled a region of the gene to be excised which coded for a mobile surface loop of polypeptide (residues 98-110) which normally seals the active site vacuole from bulk solvent and is a major determinant of substrate specificity. Oligonucleotide-overlap extension (using the polymerase chain reaction) was used to obtain double-stranded DNA regions which coded for different length and sequence loops and which also contained the same restriction sites. The variable length and sequence loops were inserted into the cut gene and used to synthesize hydroxyacid dehydrogenases with altered substrate specificities. Loops which were longer and shorter than the original were made. The substrate specificities of enzymes with these new loops were considerably altered. For many poor enzyme-substrate pairs, the effect of fructose 1,6-bisphosphate on the steady-state kinetic parameters suggested that the substrate was mainly bound in a nonproductive mode. With one longer loop construction (BL1), activity with pyruvate was reduced one-million-fold but activity with phenylpyruvate was largely unaltered. A switch in specificity (kcat/KM) of 390,000-fold was achieved. The 1700:1 selectivity of enzyme BL1 for phenylpyruvate over pyruvate is that required in a phenyllactate dehydrogenase to be used in monitoring phenylpyruvate in the urine of patients with phenylketonuria consuming an apparently phenylalanine-free diet.

Amino Acid Sequence↗

PubMed1973

Aminoacid sequence of dogfish M4 lactate dehydrogenase.

About 80% of the aminoacid sequence of dogfish (Squalus acanthius) M(4) lactate dehydrogenase (EC 1.1.1.27) has been elucidated. Several sequence homologies with peptides from pig H(4) and pig M(4) lactate dehydrogenase are identified. Histidine 195 is homologous to the essential histidine residue in pig H(4) lactate dehydrogenase. Similarities in the sequence around the "essential" cysteine residue of lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and yeast and liver alcohol dehydrogenase are delineated.

Amino Acid Sequence↗

PubMed1981

[Immunochemical study of lactate dehydrogenase polymorphism in the loach Misgurnus fossilis].

Data from experiments on antiserum against B4-isoenzyme of lactate dehydrogenase confirmed the previously made assumption that the main isoenzyme of lactate dehydrogenase of most differentiated tissues of the loach Misgurnus fossilis, which is inhibited by AgNO3, is made up by A-subunits, while that of the isoenzymes whose activity is predominant in the ovaries and embryos of this teleost species and which are normally resistant to AgNO3, are made up by B-subunits. The C4-isoenzyme of lactate dehydrogenase which is liver-specific is highly sensitive to inactivation by anti-B4 antiserum. The "minor" isoforms of the enzyme detected in the enzymatic spectrum of the ovaries, embryos and larvae, which are inhibited by AgNO3, are subjected to inactivation by anti-B4- and are insensitive to anti-A4 antiserum against A4-isoenzyme of lactate dehydrogenase from loach skeletal muscles. These data and a gradual disappearance of the isoforms simultaneously with a decrease of yolk in developing larvae suggest that they appeared due to post-translational modifications of the B-subunits, predominant in lactate dehydrogenase of ovaries and embryos by certain components of the yolk.

Animals↗