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PubMed1985

Lactate dehydrogenase isoenzymes in diabetic patients.

Lactate dehydrogenase (LDH) isoenzyme profiles in human platelets and the sera of patients with type I and II diabetes mellitus and vascular complications, as well as normal subjects were measured utilizing a recently established, modified micromethod. LDH-3 was the predominating species in platelets (37.5 +/- 3.0%), with LDH-2, 1, 4 and 5 following in decreasing order of concentration. The LDH-3/LDH-4 ratio in platelets varied from 6.2 to 1.38. Type I and type II diabetic patients with vascular complications showed a significantly higher ratio for LDH-3/LDH-4 (3.99 +/- 1.20 for DM I, 2.16 +/- 0.25 for DM II patients) than the mean ratio for normal subjects (1.14 +/- 0.08). This platelet-specific LDH isoenzyme pattern may be the result of frequent in vivo platelet-vessel wall interactions in the diabetic patients whose platelets are known to be hyperaggregable in in vitro test systems. Since non-diabetic patients patients with vascular complications also displayed a similarly elevated LDH-3/LDH-4 ratio, a wider classification is preferable, although the measurement of the LDH isoenzyme pattern will be helpful in assessing diabetic vascular complications.

Adult↗
PubMed2001

Methylation of CpG dinucleotides alters binding and silences testis-specific transcription directed by the mouse lactate dehydrogenase C promoter.

The mouse lactate dehydrogenase c gene (mldhc) is transcribed only in cells of the germinal epithelium. Cloning and analysis of the mldhc promoter revealed that a 100-base pair fragment was able to drive testis-specific transcription in vitro and in transgenic mice. Several testis-specific genes are believed to be regulated at least in part through differential methylation of CpG dinucleotides. We investigated the possibility that transcriptional repression of the mldhc gene is mediated in somatic tissues by hypermethylation of CpG dinucleotides. The CpG dinucleotides within a fragment of the mldhc promoter containing a GC box and tandem activating transcription factor/cAMP-responsive element binding sites are hypermethylated in somatic tissues and hypomethylated in testis. Methylation of the activating transcription factor/cAMP-responsive elements altered the protein binding pattern observed in electrophoretic mobility shift assays using mouse liver but not testis nuclear extract. Furthermore, methylation of an extended mldhc promoter fragment driving lac Z silenced transcription from the promoter in a transient transfection assay. These data suggest that tissue-specific differential methylation plays a role in mldhc silencing in somatic tissues.

Activating Transcription Factors↗
PubMed1978

Oxidation of sulfhydryl groups in lactate dehydrogenase under high hydrostatic pressure.

Lactate dehydrogenase from rabbit skeletal muscle in the presence of substrate exhibits irreversible deactivation at hydrostatic pressures beyond 1 kbar [Schmid, G., Lüdemann, H.-D. & Jaenicke, R. (1975) Biophys. Chem. 3, 90-98]. In the absence of substrate and coenzyme the lability towards pressure is enhanced. The pH dependence of the effect and its inhibition by SH-protecting agents suggest the oxidation of sulfhydryl groups to be involved in the mechanism of deactivation. Partial deactivation observed at pH 5.5-7.0 becomes complete in the range of the intrinsic pK of cysteine; addition of dithiothreitol and/or EDTA protects the enzyme from complete deactivation, and leads to the residual enzymatic activity observed at pH 7.0. Incubation of the enzyme with dithiothreitol after pressure deactivation at pH 8.5 causes partial reactivation. From pressure-dependent measurements of the kinetics of deactivation an activation volume of deltaVnot equal to = -285 +/- 30 cm3 . mol-1 is calculated, which exceeds numerical data reported for typical reactions in organic chemistry. Therefore, the assumption can be made that the oxidation of sulfhydryl groups is connected with structural changes in the enzyme in the rate-determining step of the deactivation. The proposed mechanism may contribute to the toxicity of oxygen towards bacteria under high hydrostatic pressure.

Animals↗
PubMed1983

Drosophila lactate dehydrogenase: developmental aspects.

Partially purified lactate dehydrogenase (LDH) from third-instar larvae displays two bands (one major and one minor) on polyacrylamide gels. Analogous preparations from pupae and adults exhibit three LDH-staining bands (one major and two minor) in a similar pattern. The migration of the major band is similar for larvae, pupae, and adults, while the two minor LDH bands of pupae and adults migrate more slowly than the minor larval band. It has been shown that larval LDH incubated with beta-nicotinamide adenine dinucleotide exhibits two additional minor bands with an electrophoretic mobility similar to that of the minor bands of both pupae and adults. The intensity of the minor larval LDH band (exhibited also by untreated preparations) is drastically reduced. This fact indicates that the life-cycle stage-dependent LDH isozymic distribution is possibly due to a posttranslational effect(s). Highly purified LDH from larvae, pupae, or adults, obtained by an affinity chromatography procedure, displays just one dispersed band, located in the area between the band 5 and the band 6 exhibited by crude extract preparations. These data, in combination with the lack of difference in catalytic properties among enzymes from larvae, pupae, and adults, suggest that LDH synthesis is controlled by the same single structural gene at all developmental stages.

Aging↗
PubMed1983

Characterization of monoclonal antibodies to the sperm-specific lactate dehydrogenase isozyme.

The isozyme of lactate dehydrogenase (LDH) that is specific to testes, designated LDH-C4, is the predominant LDH isozyme in mammalian spermatozoa. Four high-affinity monoclonal antibodies have been developed to murine LDH-C4. These antibodies were tested for crossreactivity with LDH-C4 from rat, hamster, rabbit, and human by competitive binding radioimmunoassays. Monoclonal antibodies RG-1 and RG-2 are specific for adjacent or partially overlapping epitopes. The other two monoclonal antibodies each recognize separate and distinct determinants. One of these, designated RG-4, recognizes a sequential determinant that is contained in the coenzyme binding loop, residues 101-115 of the C subunit. Furthermore, RG-4 shows reduced binding affinity for rat LDH-C4 which differs in amino acid sequence at residue 108 and 111 in this region of the molecule. RG-4 also has reduced affinity for LDH-C4 of other species, which is consistent with substitutions in the amino acid sequences of the coenzyme binding loop. These differences between C4 of closely related species is in contrast to the high degree of conservation of this sequence in the LDH-A4 and LDH-B4 isozymes. These results provide useful information regarding homologies among species of LDH-C4 as well as the evolution of this isozyme.

Animals↗
PubMed1989

Molecular properties of pyruvate bound to lactate dehydrogenase: a Raman spectroscopic study.

Lactate dehydrogenase (LDH; EC 1.1.1.27) catalyzes the addition of pyruvate to the four position of the nicotinamide ring of bound NAD+; this NAD-pyruvate adduct is bound tightly to the enzyme. We have used the adduct as a model for pyruvate in a competent ternary complex by comparing the Raman spectrum of the bound adduct with that for unliganded pyruvate. To understand the observed normal modes of pyruvate both as the bound adduct and in water, we have taken the Raman spectra of a series of 13C- and 18O-labeled pyruvates. We find that the carboxylate COO- moiety of pyruvate remains unprotonated at LDH's active site and forms an ion pair complex. The frequency of pyruvate's carbonyl C = O moiety shifts from 1710 cm-1 in water downward 34 cm-1 when pyruvate binds to LDH. This frequency shift corresponds to a ca. 34% polarization of the carbonyl bond, indicates a substantial interaction between the C = O group and enzyme, and is direct evidence for and is a measure of enzyme-induced electronic perturbation of the substrate needed for catalysis. This bond polarization is likely brought about by electrostatic interactions between the carbonyl moiety and the protonated imidazole group of His-195 and the guanidino group from Arg-109. We discuss how the data bear on the enzymatic chemistry of LDH.

Binding Sites↗
PubMed1973

Reconstitution of D-lactate-dependent transport in membrane vesicles from a D-lactate dehydrogenase mutant of Escherichia coli.

Membrane-bound, flavin-linked D-lactate dehydrogenase in membrane vesicles of E. coli ML 308-225 is solubilized by extraction with guanidine HCl. When membrane vesicles prepared from a D-lactate dehydrogenase mutant are treated with this extract, they regain the capacity to catalyze D-lactate oxidation and D-lactate-dependent transport. Similar effects are obtained with wild-type membrane vesicles in which D-lactate oxidation and D-lactate-dependent transport have been inactivated by 2-hydroxy-3-butynoate. Although treatment of wild-type vesicles with the extract results in an increased capacity to catalyze D-lactate oxidation, no effect on transport is observed. Reconstituted transport activity is a saturable function of the amount of guanidine extract added. Moreover, the quantity of extract required to achieve maximum initial rates of transport varies with each transport system. On the other hand, reconstituted D-lactate oxidation increases linearly over a broader range of extract concentrations.Oxamate, a competitive inhibitor of D-lactate dehydrogenase, and p-chloromercuribenzenesulfonate block both the initial rate of transport and the steady-state level of accumulation in reconstituted vesicles. Furthermore, these reagents induce efflux of transport substrates from preloaded, reconstituted vesicles. The same reagents inhibit the initial rate of uptake but not the steady-state level of accumulation in ML 308-225 vesicles, and do not induce efflux. These results suggest that, although reconstituted vesicles catalyze D-lactate oxidation and D-lactate-dependent transport, the system has not been reconstituted to its native state.

Amino Acids↗
PubMed2006

Expression of vascular endothelial growth factor, fibroblast growth factor, and lactate dehydrogenase by human peritoneal mesothelial cells in solutions with lactate or bicarbonate or both.

In patients on long-term continuous ambulatory peritoneal dialysis, the efficiency of dialysis declines because of peritoneal neovascularization and loss of peritoneal mesothelial cells. In this study, we investigated the influence of lactate and bicarbonate in peritoneal dialysis fluid on such changes of the peritoneum. We studied the production of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which induce peritoneal neovascularization, by human peritoneal mesothelial cells (HPMCs) cultured with lactate or bicarbonate or both. Lactate dehydrogenase (LDH) was also measured to assess cell necrosis. Levels of VEGF, bFGF, and LDH in the culture supernatant showed a significant decrease after incubation of HPMCs with 15 mEq/L lactate plus 25 mEq/L bicarbonate, or with 40 mEq/L bicarbonate, as compared with incubation with 40 mEq/L lactate. Levels of VEGF and bFGF showed a concentration-dependent decrease when the cells were incubated with lactate or bicarbonate; a concentration-dependent increase of LDH was simultaneously observed. These results suggest that dialysis fluid containing 40 mEq/L bicarbonate is superior to fluid containing 40 mEq/L lactate with regard to its influence on the production of VEGF and bFGF although lactate and bicarbonate are both toxic for HPMCs.

Bicarbonates↗
PubMed1984

Structural comparison of lactate dehydrogenase homologs differing in sensitivity to hydrostatic pressure.

The muscle-type (M4) lactate dehydrogenases (L-lactate: NAD+ oxidoreductase EC 1.1.1.27) of two teleost fishes, Sebastolobus alascanus and Sebastolobus altivelis , differ in the susceptibility of ligand binding to perturbation by moderate hydrostatic pressures. The enzyme homologs were purified by affinity chromatography. The amino-acid compositions of these enzymes are virtually identical. The proteins were digested with trypsin and the peptide mixtures mapped using reverse-phase HPLC. Although there was variation in elution times of some peaks, the amino-acid compositions of the fractions from the two profiles were highly similar. Only one clear difference in amino-acid composition was found and this peptide was sequenced using the manual dansyl-Edman method. The enzyme of S. alascanus , which is susceptible to pressure-perturbation, had a histidine at position 115; the S. altivelis enzyme had an asparagine. Ionization of histidine is affected by pressure and may be involved in the differences between the two lactate dehydrogenase homologs. There is no covalently bound phosphate associated with either enzyme, and thus phosphorylation cannot account for the differences between the enzyme homologs. Acquisition of pressure-tolerance appears to involve only minor changes in primary structure.

Amino Acid Sequence↗
PubMed1983

A quantitative histochemical study of lactate dehydrogenase and succinate dehydrogenase activities in the membrana granulosa of the ovulatory follicle of the rat.

Using a microdensitometer, lactate dehydrogenase and succinate dehydrogenase activities were measured in the membrana granulosa of the rat ovulatory follicle. Ovaries were removed on each day of the oestrous cycle; oestrus, dioestrus-1, dioestrus-2, and proestrus; and enzyme activities measured in the membrana granulosa as a whole and in four regions within it: peripheral (PR), antral (AR), cumulus oophorus (CO) and corona radiata (CR). Throughout the cycle, lactate dehydrogenase activity was greatest in PR. On oestrus, lactate dehydrogenase activity was progressively less in AR, CO and CR. On dioestrus-1, activity was identical in AR and CO and less in CR. On dioestrus-2, activity was greater in AR than in CO or CR. By proestrus, activity was equal in AR, CO and CR. In the membrana granulosa as a whole, and in each region, lactate dehydrogenase activity declined as ovulation approached. In contrast, succinate dehydrogenase activity in the membrana granulosa as a whole and in PR was constant throughout the cycle. Activity fluctuated in the other regions. Succinate dehydrogenase activity on oestrus was greatest in PR, less in AR and CO and least in CR. On the remaining days, succinate dehydrogenase activity was greatest in PR and less but equal in the remainder of the membrana granulosa.

Animals↗
PubMed1975

Pretreatment serum lactate dehydrogenase predicting metastatic spread in Ewing's sarcoma.

We measured serum lactate dehydrogenase levels in 36 patients with localized Ewing's sarcoma before treatment. The results were evaluated to determine if the levels served as a prognostic indicator for metastatic spread. After adjusting for primary site of disease and adjuvant chemotherapy, the pretreatment serum lactate dehydrogenase proved an extremely good predictor of which patients would ultimately develop metastatic disease. The median serum lactate dehydrogenase of the total group was between 201 and 214 IU/litre. Three of 18 patients presenting with serum levels of this enzyme of less than or equal to 201 IU/litre ultimately developed metastases, while 16 of 18 patients who presented with serum levels of greater than or equal to 214 IU/litre developed metastases (P less than 0.001).

Cyclophosphamide↗
PubMed1996

Functional properties of the histidine-aspartate ion pair of flavocytochrome b2 (L-lactate dehydrogenase): substitution of Asp282 with asparagine.

The FMN prosthetic group of flavocytochrome b2 or L-lactate dehydrogenase oxidizes lactate to pyruvate. The reducing equivalents are then transferred one by one, intramolecularly, to heme b2 and then to external acceptors. Substrate oxidation is thought to begin with abstraction of the substrate alpha-hydrogen as a proton by an enzyme base. It has been proposed that this role is played by His373, which lies close to the flavin in the crystal structure and interacts with Asp282. It has also been shown before, using hydrogen exchange measurements, that the pKa of His373 is substantially increased in the wild-type reduced enzyme compared to that in the oxidized state. We report here the enzymatic properties of the D282N mutant flavocytochrome b2. Steady-state rate measurements with [2-1H]lactate and [2-2H]-lactate indicate that, as predicted, the Michaelis complex stability is hardly affected, whereas the transition state for proton abstraction increases in energy by 2.8 kcal/mol. Steady-state inhibition studies were conducted with a number of active-site ligands: sulfite, D-lactate, pyruvate, and oxalate. Binding was found to be most affected for oxalate, but kinetic patterns indicated oxalate and pyruvate were still capable of binding to the enzyme both at the oxidized and semiquinone stages, whereas inhibition by excess substrate, due to lactate binding at the semiquinone stage, was lost. Finally, analysis of the intermolecular hydrogen transfer catalyzed by the enzyme between [2-3H]lactate and fluoropyruvate indicated that the substitution with asparagine facilitates exchange of the histidine-bound proton and hence induces a decrease in the pKa value of H373 in the reduced enzyme of about 1.4 pH units. Nevertheless, the rate constant value for exchange with the solvent of the enzyme-bound substrate alpha-proton indicates that H373 is still protonated in the reduced mutant enzyme at neutral pH. Thus, the D282N mutation destabilizes the transition state for proton abstraction and decreases the pKa of H373 in the reduced enzyme but is insufficient to bring it back to a normal value.

Asparagine↗
PubMedDate not supplied

[Active forms of oxygen and the degree of UV modification of the structural and functional properties of lactate dehydrogenase].

The spectrophotometry and photofluorescence techniques were used in the studies on photochemical transformations of lactate dehydrogenase exposed to UV irradiation with a dose of 2.25 kJ/m2, in the native state and in the presence of exogenous modifiers: sodium azide, beta-carotene, histidine, D-mannitol, and tret-butanol. It was shown that UV irradiation of the mixtures of lactate dehydrogenase with sodium azide, beta-carotene, and histidine results in restoration (by 99, 65, and 63%, respectively) of the level of catalytic activity of the enzyme as compared to that observed after irradiating it in the absence of the protectors. The protective effect provided by mannitol during UV irradiation of the lactate dehydrogenase was 23%. Thus, it was shown that active oxygen species--singlet molecular oxygen and hydroxyl radical--make significant contributions to photomodification of lactate dehydrogenase.

Hydrogen-Ion Concentration↗
PubMed1972

Genetic control of lactate dehydrogenase expression in mammalian tissues.

The amount of lactate dehydrogenase isozyme 4 in erythrocytes of mice is controlled by alleles at the Ldr-1 locus. The A subunits of lactate dehydrogenase from erythrocytes deficient in isozyme 4 cannot assemble in vitro with B subunits to form active isozyme. The inability to form hybrid enzyme is not due to a mutation in the structural gene for the A polypeptide. Rather, a factor that is bound to the A subunits of erythrocytes restricts free exchange with B subunits.

Animals↗
PubMed1975

Immunochemical properties of the membrane-bound D-lactate dehydrogenase from Escherichia coli.

The preparation, characterization, and purification of antibody against the membrane-bound D-lactate dehydrogenase solubilized and purified from Escherichia coli ML 308-225 are described. The antibody is highly specific for the flavin-linked D-lactate dehydrogenase, and incubation of the enzyme with antiserum results in marked inhibition of enzymatic activity. By means of a radioimmune assay, it is demonstrated that membrane vesicles prepared from E. coli ML 308-225dld-3 contain catalytically inactive material which cross-reacts with native D-lactate dehydrogenase. In the following paper, the effects of this antiserum on D-lactate dehydrogenase activity and D-lactate-dependent active transport in native and reconstituted membrane vesicles are examined.

Animals↗