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PubMed1998

Histochemical determination of lactate dehydrogenase activity in rat lens; influence of different parameters.

PURPOSE: 1. To optimize a histochemical technique for the determination of lactate dehydrogenase activity in rat lens. 2. To determine the distribution of lactate dehydrogenase activity within the lens. 3. To analyse different components of variation in the method. METHODS: A total of 24 six-week-old Sprague-Dawley rats were used. Cryosections of whole eyes were incubated with different media. The influence of incubation time, pH, the concentration of dye, lactate, nicotinamide dinucleotide, phenazine methosulfate, sodium azide, hydrazine and polyvinyl alcohol on the enzyme histochemical reaction was investigated. The staining reactant was nitrobluetetrazolium and the staining density was measured with a microscope-based photometer. RESULTS: The lactate dehydrogenase activity was highest in the epithelium, followed by the nucleus, the anterior cortex, and the posterior cortex. The three largest components of variation in the analyses were rats, sections (including variation in section thickness) and density measurements (including variation within the different regions). CONCLUSION: The composition of the final incubation medium for the detection of lactate dehydrogenase activity in rat lens are similar to that shown for other tissues. Lactate dehydrogenase activity varies in different regions of the lens.

Animals↗
PubMed1995

Purification and properties of lactate dehydrogenase from liver of Uromastix hardwickii.

Lactate dehydrogenase isoenzyme-1 was purified from liver of Uromastix hardwickii using colchicine-Sepharose and heat-inactivation methods. The crude enzyme showed four isoenzymes by agarose gel electrophoresis (AGE). The purified enzyme showed a single band after native AGE and SDS-PAGE corresponding to a molecular weight of 34 kDa. The enzyme did not bind with DEAE-Sepharose at pH 7.2. The optimum pH for forward reaction was 7.5, while for reverse reaction, the maximum activity was at pH 9.5. The Km values for pyruvate, NADH, lactate and NAD+ were 0.105, 0.045, 9.0 and 0.011 mM, respectively. The pyruvate showed maximum activity at about 150 microM and then starts showing inhibition at higher concentration. Pre-heating of enzyme showed that it was stable at 80 degrees C for 30 min and at 100 degrees C it became inactive immediately. Oxalate, glutamate, Cu2+, Co2+, Mn2+, and Mg2+ have shown inhibitory effects both for forward- and reverse-reactions. From these properties, we suggest that LDH-1 from Uromastix liver may be quite different from that of other vertebrates.

Animals↗
PubMed1994

Effect of tubulin on the activity of the muscle isoenzyme of lactate dehydrogenase.

The interaction between muscle-type lactate dehydrogenase (LDHm) and tubulin was investigated by monitoring the combined effect of NADH and tubulin on steady-state kinetics and the combined effect of NADH and pH on complex formation between tubulin and the enzyme. Steady-state kinetics showed that LDHm is inhibited by tubulin. Experiments with heart-type lactate dehydrogenase (LDHh) showed that the inhibition is unique to the muscle-type enzyme. The magnitude of the inhibition is dependent upon the concentration of NADH as well as the pH of the buffer medium. The enzyme was less sensitive to inhibition at 50 microM NADH than at 10 microM NADH. Since this effect of NADH is not due to an ionic strength contribution, it is deemed to be specific. In contrast to the absence of tubulin, its presence induced a modification of the kinetic behavior of LDHm; i.e., the velocity dependence on NADH concentration displayed a marked sigmoid response. The inhibition of LDHm by tubulin is more pronounced at lower pH values than at higher pH values. The pH-dependent inhibitory profile is shifted to the left (i.e., pKa is decreased) with increasing concentrations of NADH. This pattern is remarkably similar to that observed for the binding of the enzyme on Sepharose immobilized tubulin and is consistent with the premise that inhibition is a result of interaction between these proteins. NAD+ was much less effective than NADH in dissociating LDHm from immobilized tubulin. Results from these in vitro studies are consistent with similar observations dealing with other glycolytic enzymes and cytoskeleton proteins, which show that enzyme catalytic properties are modified upon binding.

Animals↗
PubMed1979

A detailed investigation of the properties of lactate dehydrogenase in which the 'Essential' cysteine-165 is modified by thioalkylation.

The reaction of pig heart lactate dehydrogenase with methyl methanethiosulphonate resulted in the modification of one thiol group per protomer, and this was located at cysteine-165 in the enzyme sequence. On reduction, both the thiomethylation of cysteine-165 and any changes in kinetic properties of the enzyme were completely reversed. Cysteine-165 has been considered essential for catalytic activity; however, cysteine-165-thiomethylated dehydrogenase possessed full catalytic activity, although the affinity of the enzyme for carbonyl-or hydroxy-containing substrates was markedly decreased. The nicotinamide nucleotide-binding capacity was unaffected, as judged by the formation of fluorescent complexes with NADH. The enzyme-mediated activation of NAD+, as judged by sulphite addition, was unaffected in thiomethylated lactate dehydrogenase. However, the affinity of oxamate for the enzyme--NADH complex was decreased by 100-fold and it was calculated that this constituted a net increase of 10.4 kJ/mol in the activation energy for binding. Thiomethylated lactate dehydrogenase was able to form an abortive adduct between NAD+ and fluoropyruvate. However, the equilibrium constant for adduct formation between pyruvate and NAD+ was too low to demonstrate this complex at reasonable pyruvate concentrations. A conformational change in the protein structure on selective thiomethylation was revealed by the decreased thermostability of the modified enzyme. The alteration of lactate dehydrogenase catalytic properties on modification depended on the bulk of the reagent used, since thioethylation resulted in an increase in Km for pyruvate (13.5 +/- 3.5 mm) and an 85% decrease in maximum catalytic activity. The implications of all these findings for the catalytic mechanism of lactate dehydrogenase are discussed.

Alkylation↗
PubMed2002

Purification and kinetic properties of skeletal muscle lactate dehydrogenase from the lizard Agama stellio stellio.

Lactate dehydrogenase isoenzyme LDH-5 (M4) was purified to homogeneity from the skeletal muscle of lizard Agama stellio stellio as a poikilothermic animal, using colchicine-Sepharose chromatography and heat inactivation. The purified enzyme showed a single band after SDS-PAGE, corresponding to a molecular weight of 36 kD. The Km values for pyruvate, NADH, lactate, and NAD+ were 0.020, 0.040, 8.1, and 0.02 mM, respectively. Pyruvate showed maximum activity at about 180 microM, with a decline at higher concentrations. The enzyme was stable at 70 degrees C for 30 min, but was rapidly inactivated at 90 degrees C. The optimum pH for the forward reaction (pyruvate to lactate) was 7.5, and for the reverse reaction (lactate to pyruvate) was 9.2. Oxalate, glutamate, Cu2+, Co2+, Mn2+, and Mg2+ were inhibitory in both forward and reverse reactions.

Animals↗
PubMed1986

Serum lactate dehydrogenase level in various malignancies.

Total serum lactate dehydrogenase (SLD) values were measured and statistically analyzed with a view to testing the hypothesis that there is some association between enzymatic activity and cancer. Serum samples were collected from 205 normal persons, 201 patients with different malignant tumors and 202 patients with nonmalignant diseases. The age of patients and controls ranged from 19 to 71 years. There is a significant rise in preoperative serum lactate dehydrogenase level in cancer patients (314.56 U/l) than normal healthy individuals (105.50 U/l) and nonmalignant patients (105.87 U/l) irrespective of the site of tumor. The patient and control series did not show any significant bisexual differences. The postoperative follow-up study (breast cancer only) of SLD level indicated decreased enzymatic activity in almost all the cases. The observations of elevation of SLD in patient series than controls suggest release of LD to the surrounding medium by neoplastic cells, thereby indicating relationship between the enzymatic activity and neoplastic conditions. The results of preoperative and postoperative follow-up study of SLD activity in breast cancer prove of value in judging the effectiveness of therapy, diagnosis and prognosis of the cancer patients.

Breast Neoplasms↗
PubMed1977

Enzymatic activity patterns in probability population samples. Serum lactate dehydrogenase.

Sex- and age-specific serum lactate dehydrogenase values provided by a natural population of 2,378 individuals selected by a multistage area cluster probability sampling scheme are given. The activity of this enzyme has definite sex and age associations over the age range studied (larger than or equal to 10 years). In both sexes, it is characterized by elevations in preadolescents, by a sharp decrease in adolescent groups, and by a gradual increase in adult and senescent groups, which, however, does not bring it back to earlier levels. Males have higher mean serum lactate dehydrogenase activities than females in most age groups until about 50 years of age. Unusually high activities have been found in this study, compared with those found in two other widely separated areas in North America using the same assay procedures.

Adolescent↗
PubMed1979

Lactate-to-pyruvate or pyruvate-to-lactate assay for lactate dehydrogenase: a re-examination.

The pyruvate-to-lactate assay for determining lactate dehydrogenase (EC 1.1.1.27) can now yield linearity equal to or better than that obtained by the lactate-to-pyruvate assay. In addition, there are significant advantages to the pyruvate-to-lactate reaction: (a) a greater change in absorbance per unit time, which allows more accurate spectrophotometric readout; (b) lower reactant concentrations are required, which substantially reduces the cost per assay; (c) solid reagents are used to prepare the assay solution; and (d) reagent solutions are more stable. However, impurities present in commercial NADH preparations may substantially affect measured lactated dehydrogenase activities; therefore, a Standard Reference Material for NADH is being developed for issuance by the National Bureau of Standards.

L-Lactate Dehydrogenase↗
PubMed1987

The primary structure of the lactate dehydrogenase isozyme M4 from giant panda.

The amino acid sequences of tryptic, chymotryptic and cyanogen bromide cleavage peptides of the lactate dehydrogenase isozyme M4 from giant panda have been determined. Based on the overlapping peptides and by a comparison with the known sequence of porcine lactate dehydrogenase isozyme M subunit, the complete primary structure of the giant panda lactate dehydrogenase isozyme M subunit has been established. The polypeptide chain of giant panda lactate dehydrogenase isozyme M subunit consists of 331 amino acid residues. There is a variance of 17 amino acid residues between the porcine and the giant panda lactate dehydrogenase isozyme M subunits. Most of the variable residues are substitutions by chemically similar amino acids and take place at residues not related to the active center of the enzyme.

Amino Acid Sequence↗
PubMed2004

Effect of selected alcohol dehydrogenase inhibitors on the human heart lactate dehydrogenase activity--an in vitro study.

Metabolic acidosis complicates methanol, ethylene glycol and other alcohol intoxications. It is caused firstly by acid metabolites and secondly by the lactate elevation. The aim of the study was to evaluate the effect of alcohol dehydrogenase (ADH; EC 1.1.1.1) inhibitors and substrates: 4-methylpyrazole (4-MP), cimetidine, EDTA, ethanol and methanol on lactate dehydrogenase (LDH; EC 1.1.1.27) activity. The activity of LDH was determined spectrophotometrically in in vitro human heart homogenates with the mentioned compounds at 0.01, 0.1, 1.0 mM concentrations of 4-MP, cimetidine, EDTA, and 12.5, 25.0, 50.0 mM of ethanol and methanol. The LDH activity was significantly inhibited by 0.1 mM (p<0.05) and 1.0 mM (p<0.01) 4-MP and 1.00 mM EDTA (p<0.05). Higher LDH activity vs. control was observed in the samples incubated with all studied ethanol and methanol concentrations but these differences were not statistically significant. Thus, 4-MP was found to be the most effective inhibitor of LDH of all compounds tested. Therefore, such effect of 4-MP seems to be an additional advantage in methanol and ethylene glycol intoxications.

Adult↗
PubMed1971

Estimation of the growth of the T 1 strain of Mycoplasma mycoides in tryptose broth by the measurement of lactate dehydrogenase. I. Method.

A highly significant correlation coefficient (r = 0.97, n = 18) was found between the concentration of lactate dehydrogenase measurable after the organisms had been disrupted and the concentration of colony-forming units during the logarithmic phase of growth of a broth culture of the T(1) strain of Mycoplasma mycoides var. mycoides. A concentration of 4.60 x 10(-7) milliunits of lactate dehydrogenase for each colony-forming unit was established. This relationship was used to convert the concentration of lactate dehydrogenase in the culture into an estimate of the concentration of viable mycoplasma. The lactate dehydrogenase was estimated by following the oxidation of reduced nicotinamide adenine dinucleotide, in the presence of pyruvate substrate, at 366 nm in a spectrophotometer. The nicotinamide adenine dinucleotide oxidase system probably contributed a small amount of enzyme activity to the test when lactate dehydrogenase was measured in this way. The method has been described and evaluated for the estimation of titers from 10(7) to 5 x 10(9) colony-forming units per ml.

Bacteriological Techniques↗
PubMed1979

The effects of season and temperature on D-lactate dehydrogenase, pyruvate kinase and arginine kinase in the foot of Helix pomatia L.

The effects of pH, season, environmental and experimental temperatures on the activities and kinetic parameters of D-lactate dehydrogenase, pyruvate kinase and arginine kinase from the foot of the pulmonate snail Helix pomatia were analyzed. Both in phosphate and Tris buffers D-lactate dehydrogenase was the enzyme with the most acid maximum, arginine kinase that with the most alkaline, whilst pyruvate kinase occupied an intermediate position. Pyruvate kinase activity, measured at 20 degrees C, was positively correlated with the environmental temperature at the moment of collecting the animal, whereas neither arginine kinase nor D-lactate dehydrogenase showed such a relationship. A seasonal study based on approximately 100 specimens established that arginine kinase activity remained the same throughout the year. Pyruvate kinase activity was slightly lower, and D-lactate dehydrogenase activity significantly higher, in winter than in summer animals. Snails subjected in spring to a short warm-up period before enzyme extraction showed extreme variability and some extraordinarily high values of pyruvate kinase activity, suggesting that either season or elevated temperature may have an immediate effect on the activity of this enzyme. Individual variability of all three enzymes ranges from 300 to 400%. The activities of pyruvate kinase and D-lactate dehydrogenase are strongly correlated in summer, forming a "constant-proportion-group", whereas in winter, with D-lactate dehydrogenase activity increasing and pyruvate kinase activity decreasing these two enzymes become "uncoupled". The Km value of pyruvate kinase is independent of experimental temperature between 10 and 25 degrees C, whereas that of D-lactate dehydrogenase and arginine kinase increases about three-fold within this range. Thus the temperature relationship of a single enzymic reaction cannot be used as an arguemnt for or against the occurrence of temperature compensation of whole animal metabolism. The possibility of modulation of enzyme activity by environmental temperature is discussed.

Acclimatization↗
PubMed1983

A study of lactate dehydrogenase levels and turnover rates during postnatal development in the rat.

Specific radioimmunoassays for lactate dehydrogenase A and B subunits have been employed to quantify cellular contents of these proteins more precisely than hitherto possible and to monitor changes during postnatal development. Liver, skeletal muscle, heart muscle and kidney cortex all demonstrated alterations in cellular levels of lactate dehydrogenase subunits over the first 56 days of life, the particular pattern being specific to each tissue. Studies on the turnover of lactate dehydrogenase in vivo and in vitro indicated that the developmental changes in total lactate dehydrogenase content in liver and kidney were regulated at some point(s) during both the biosynthesis and the degradation of the proteins.

Aging↗
PubMed1985

Kinetic analysis of lactate dehydrogenase in cultured chondrocytes by quantitative cytochemistry.

Kinetic analysis of lactate dehydrogenase activity in intact cultured chondrocytes was performed in situ by coupling cell culture and microcytophotometry. Cells were cultured on glass microscope slides divided into eight chambers and studied during the growth cycle in monolayer areas. Lactate dehydrogenase activity was assayed by the reduction of neotetrazolium in the presence of phenazine methosulfate. Quantification of formazan deposits within the cells was performed by scanning and integrating microdensitometry at the isosbestic wavelength of 585 nm. Results indicate the following (a) A kinetic characterization was possible: apparent constants, Km and Ks of this two-substrate enzyme were graphically determined Ks = 1.05 +/- 0.08 and 0.56 +/- 0.05 mM for lactate and NAD respectively and Km = 0.64 +/- 0.03 and 0.37 +/- 0.02 mM for lactate and NAD respectively. (b) Inhibition by lactate concentrations above 10 mM and pyruvate concentration of 1 mM, is in agreement with the well known high anaerobic glycolytic metabolism of chondrocytes. This was confirmed by electrophoresis on cellulose acetate which demonstrated a M3-H isoenzyme form in cultured chick chondrocytes. This study shows that microcytophotometric analysis of lactate dehydrogenase in cultured chondrocytes may be an interesting alternative to mass culture cells followed by classical biochemical studies.

Animals↗
PubMed1984

On the mechanism of lactate dehydrogenase leakage from normal and D-galactosamine-treated hepatocytes in monolayer culture.

Synthesis, degradation and leakage of lactate dehydrogenase and of total protein was measured using D-galactosamine-treated rat hepatocytes in monolayer culture. The kinetics of [3H]leucine incorporation into trichloroacetic acid-precipitable material and into isolated lactate dehydrogenase of cells and of the extra-cellular space revealed a similar extent of inhibition of both synthesis and leakage following exposure to D-galactosamine. Hepatocyte cultures that had been labeled before D-galactosamine treatment lost intracellular protein-associated radioactivity almost as rapidly as control cells up to the time of measurable enzyme leakage; thereafter, the rate of 3H-loss increased in the treated cells. Lactate dehydrogenase present in the medium is degraded less rapidly than the enzyme in the intracellular space. This explains the apparent increase of total lactate dehydrogenase activity in D-galactosamine-treated as compared to control cultures. Following [3H]leucine addition to D-galactosamine-treated cultures, the specific radioactivity of the leaked lactate dehydrogenase in the medium was never greater than that of the enzyme in the cytosolic compartment. The data rule out a direct excretion of newly synthesized enzyme as a result of D-galactosamine action.

Animals↗