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PubMed1977

[Changes in the activity of succinate dehydrogenase and lactate dehydrogenase in the muscles of patients with progressive muscular dystrophy].

Histochemical investigation of 32 biopsy specimens of the muscular tissue taken in patients with Erb's myopathy and 15 bioptic materials from patients with Charcot--Marie--Tooth's amyotrophy was carried out. Characteristics of distribution of enzymes (succinate dehydrogenase, lactate dehydrogenase) in the muscular tissue of the patients referred to above are presented. A quantitative evaluation of the activity of enzymes was made by the method of count of granules of diphormazan with subsequent treatment of data by the statistical variation method.

Histocytochemistry↗

PubMed1981

Cyclic AMP regulation of lactate dehydrogenase. Quantitation of lactate dehydrogenase M-subunit messenger RNA in isoproterenol-and N6,O2'-dibutyryl cyclic AMP-stimulated rat C6 glioma cells by hybridization analysis using a cloned cDNA probe.

We have cloned DNA complementary to mRNA coding for rat C6 glioma cell lactate dehydrogenase M-subunit. Double-stranded DNA complementary to a portion of lactate dehydrogenase mRNA was inserted into the Pst I site of plasmid pBR322 by the dC.dG tailing technique and amplified in Escherichia coli HB101. A recombinant plasmid containing lactate dehydrogenase cDNA was identified by colony hybridization to a cDNA prepared from partially purified lactate dehydrogenase mRNA and by hybridization-selected translation. The recombinant plasmid (pRLD42) contains a 680 nucleotide insert of lactate dehydrogenase mRNA. Hybridization of nick-translation pRLD42 to glioma cell poly(A)+RNA separated on agarose gel and transferred to nitrocellulose exhibited Mr = 5.9 X 10(5) for lactate dehydrogenase mRNA. Furthermore, Northern blot analysis of RNA from unstimulated and isoproterenol-stimulated glioma cells indicated a 2-fold increase of lactate dehydrogenase mRNA molecules in stimulated cells. The 2-fold increase of lactate dehydrogenase mRNA was confirmed by RNA-excess kinetic hybridization using pRLD42 DNA and poly(A)+RNA from unstimulated, isoproterenol-, and dibutyryl cAMP-stimulated glioma cells. These data demonstrate that isoproterenol and dibutyryl cAMP cause an increase of the number of lactate dehydrogenase M-subunit mRNA molecules in glioma cells which, in part, determines the extent of synthesis of the lactate dehydrogenase M-subunit.

Animals↗

PubMed1999

Ability of cytosolic malate dehydrogenase and lactate dehydrogenase to increase the ratio of NADPH to NADH oxidation by cytosolic glycerol-3-phosphate dehydrogenase.

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, the Vmax of the NADPH oxidation is only slightly lower than the Vmax of NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 microM) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 microM NADH but had no effect on oxidation of 20 microM NADPH by glycerol-3-phosphate dehydrogenase. Consequently malate dehydrogenase increased the ratio of NADPH to NADH oxidation of glycerol-3-phosphate dehydrogenase. On the basis of the measured KD of complexes between malate dehydrogenase and these reduced pyridine nucleotides, and their Km in the glycerol-3-phosphate dehydrogenase reactions, it could be concluded that malate dehydrogenase would have markedly inhibited NADPH oxidation and inhibited NADH oxidation considerably more than observed if its only effect were to decrease the level of free NADH or NADPH. This indicates that due to the opposite chiral specificity of the two enzymes with respect to reduced pyridine nucleotides, complexes between malate dehydrogenase and NADH or NADPH can function as substrates for glycerol-3-phosphate dehydrogenase, but the complex with NADH is less active than free NADH, while the complex with NADPH is as active as free NADPH. Mg2+ enhanced the interactions between malate dehydrogenase and glycerol-3-phosphate dehydrogenase described above. Lactate dehydrogenase (E.C. 1.1.1.27) had effects similar to those of malate dehydrogenase only in the presence of Mg2+. In the absence of Mg2+, there was no evidence of interaction between lactate dehydrogenase and glycerol-3-phosphate dehydrogenase.

Animals↗

PubMed1999

Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase.

Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently.

Amino Acid Sequence↗

PubMed1986

Effect of age on the crystalline rat liver lactate dehydrogenase.

Lactate dehydrogenase (LDH) was purified and crystallised from the liver of immature (4 weeks), young (22 weeks) and old (116 weeks) female rats and the kinetic, physical and chemical properties of the purified enzyme were studied. Even though slight differences existed in effect of pH, temperature, storage and ratio of A280/260, significant differences were not observed in kinetic properties, behaviour on polyacrylamide gels, ion-exchange chromatography column, and -SH groups among the three age groups. These data indicate that, unlike muscle LDH of old rats which had shown altered properties (earlier report from our laboratory), liver LDH is an unaltered, like LDH of heart. The possible reason for the unaltered nature of the liver LDH may be: (a) the tissue origin; (b) the gene responsible for its synthesis is expressing well even in old age; or (c) non-influence of post-translational modifications on the enzyme molecule.

Age Factors↗

PubMed1990

Existence of three forms of H2M2 isoenzyme of lactate dehydrogenase.

Lactate dehydrogenase (LDH) was purified from rat and bovine tissues by affinity chromatography on immobilized colchicine and used for the separation of isoenzymes by high-performance anion-exchange liquid chromatography (HPLC). This analysis showed the splitting of rat H2M2 into three peaks and of bovine H2M2 into two peaks. The heat stability, inactivation rate of urea and electrophoretic mobility of isoenzymes were examined and these analyses indicated differences in physicochemical properties for the respective peaks of rat and bovine H2M2. In hybridization experiments, the splitting of H2M2 into three peaks was achieved only with the combination of rat H4 and rat M4, while the other combinations of bovine H4 and bovine M4, of rat H4 and bovine M4 and of bovine H4 and rat M4 resulted in two H2M2 peaks. These results demonstrate that H2M2 of LDH in normal rat and bovine tissues is always split into two or three peaks by HPLC and that these H2M2 peaks have different physicochemical properties, suggesting the existence of three possible geometrical isomers of H2M2.

Animals↗

PubMed2005

A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase.

Lactate dehydrogenase from Bacillus stearothermophilus is specific for NAD+. There have been several attempts to alter the cofactor specificity of this enzyme, but these have yielded enzymes with relatively low activities that still largely prefer NAD+. A modified consensus approach was used to create a library of phylogenetically preferred amino acids situated near the cofactor binding site, and variants were screened for their ability to utilize NMN+. A triple mutant (Mut31) was discovered that proved to be more catalytically efficient than wild-type. Mut31 was also better at utilizing NAD+ than the wild-type enzyme and was weakly active with NADP+ and NMN+. An analysis of single amino acid substitutions suggested that all three mutations worked in a concerted fashion to yield robust cofactor utilization. When two previously identified amino acid substitutions were introduced into the Mut31 background, the resultant quintuply substituted enzyme not only utilized NADP+ far better than the wild-type enzyme, it actually inverted its preference for NAD+ and NADP+.

Amino Acid Substitution↗

PubMed1970

Biochemical properties of trypanosomatid lactate dehydrogenases.

Lactate dehydrogenase (LDH, E.C.1.1.1.27) was found in supernatant (cytoplasmic enzyme) fractions of the trypanosomatid flagellates Trypanosoma conorhini and Crithidia fasciculata if 10 mm cysteine was present in the homogenizing medium. The T. conorhini LDH activity with pyruvate as substrate was increased 35% if 5 mm cysteine was also included in reaction mixtures. K(m) values for the T. conorhini enzyme were 3.3 x 10(-4)m with pyruvate, and 1.6 x 10(-4)m with alpha-ketobutyrate. Cysteine inhibited alpha-ketobutyrate reduction. Comparison of trypanosomatid and human serum LDH enzymes with respect to K(m), substrate activity and inhibition, pH optima, and K(i) values for oxalate and oxamate indicated that the trypanosomatid isoenzymes differed significantly from serum LDH. C. fasciculata LDH was extremely labile, since 59% of the activity was lost 90 min after isolation. The role of LDH enzymes in trypanosomatid metabolism is discussed, and the results are related to other trypanosomatid LDH enzymes. The comparison of homologous enzymes in host and parasite is discussed with regard to metabolic function and a possible model system for chemotherapy.

Animals↗

PubMed1989

Effect of chemical modifications on freeze denaturation of lactate dehydrogenase.

Lactate dehydrogenase (LDH) was chemically ethyl-acetimidated (EA-), dimethyladipimidated (DMA-), carbamylated, acetylated, acetoacetylated, or succinylated in order to alter the ionic charges on the epsilon-amino group of lysine residues. Acetylation, acetoacetylation, and succinylation, which change the positive charge at the lysine side chains to a negative one, inactivated the enzymic activity, but the rest of the modifications exerted no such inactivating effects. The active modified enzymes were subjected to freeze denaturation study, using the enzymic activity as an indication of the degree of the denaturation. The active enzymes were diluted with deionized water and stored in a freezer (-23 degrees C) for 1-3 days. Enzymic activity was assayed immediately after thawing. All the modified enzymes retained their activity even after the 3-day frozen storage, while the control or native enzyme lost its activity within 1 day of storage. Furthermore, the modified LDHs freeze-stored in 0.2 M monosodium glutamate (MSG) or 0.2 M lysine-hydrochloride (Lys-HCl) retained their activity. The cryoprotective effects exerted by the modifications and by 0.2 M MSG seemed to be synergistic, whereas those exerted by the modifications and by 0.2 M Lys-HCl did not. The mechanisms of cryoprotection and freeze denaturation are discussed in relationship with the cryoprotective effect exerted by already known cryoprotectants, such as sucrose or dimethyl sulfoxide.

Animals↗

PubMedDate not supplied

Interaction of NAD-dependent dehydrogenases with human erythrocyte membranes. Evidence that D-glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase are catalytically active in a membrane-bound state.

Interaction of D-glyceraldehyde-3-phosphate dehydrogenase (GPDH) and lactate dehydrogenase with human erythrocyte membranes was studied. Under the conditions of low ionic strength, both enzymes bound to the membranes with similar affinities (kd = 1 microM). The binding was accompanied by complete inhibition of GPDH and by a 65-75% inhibition of lactate dehydrogenase (LDH). Increasing the ionic strength to physiologically meaningful values (0.15 M) completely abolished the inactivation of both dehydrogenases in the presence of erythrocyte membranes, but did not preclude their binding. These results suggest that different modes of enzyme-membrane interaction can be realized under the conditions of low and high ionic strength. They also indicate that GPDH and LDH are capable of functioning in a membrane-bound state.

Animals↗

PubMed1995

An improved spectrophotometric assay of pyruvate dehydrogenase in lactate dehydrogenase contaminated mitochondrial preparations from human skeletal muscle.

In mitochondria-enriched preparations of human skeletal muscle, the measurement of pyruvate dehydrogenase activity, as determined by conventional spectrophotometric assay of NADH accumulation, is underestimated due to the oxidizing activity of the contaminating lactate dehydrogenase. Using a model reaction system consisting of varying mixtures of purified lactate and pyruvate dehydrogenases, we found that the presence of oxamate, a competitive inhibitor of the lactate dehydrogenase, allowed the measurement of a linear rate of pyruvate dehydrogenase activity without interference from lactate dehydrogenase. In the presence of 25 mM oxamate, this holds true up to a ratio of 30:1 for lactate to pyruvate dehydrogenases, respectively. A similar result was obtained when using human skeletal muscle mitochondria contaminated by lactate dehydrogenase. Rates of pyruvate dehydrogenase activity ranging from 50 to 120 nmol/min/mg protein could be routinely measured in such mitochondrial fractions. We concluded that the use of oxamate allows a spectrophotometric assay for pyruvate dehydrogenase activity to be utilized when screening for pyruvate dehydrogenase deficiency in mitochondria-enriched preparations of human skeletal muscle.

Animals↗

PubMed1983

Glucose-6-phosphate dehydrogenase and lactate dehydrogenase activity in kangaroo and mouse oocytes.

Glucose-6-phosphate dehydrogenase and lactate dehydrogenase activity in oocytes and follicle cells of the Australian marsupial, Macropus eugenii (the tammar), and the mouse were determined by a simplified microelectrophoresis method. Mouse oocytes have approx 285 times more G6PD activity per picolitre of cytoplasm than tammar oocytes and about 10 times more LDH. The ratio of LDH to G6PD in mouse follicle cells is close to 3 whereas in the tammar it approaches unity. The very low levels of activity of G6PD in tammar oocytes may be due to transcriptional, translational or metabolic differences compared with the mouse.

Animals↗

PubMed1979

[Histochemical behavior of succinate dehydrogenase and lactate dehydrogenase as well as ribonucleic acid in the epithelium of lactic ducts and alveoli of cow udder].

The activities of succinate dehydrogenase (SDR) and lactate dehydrogenase (LDH) as well as the ribonucleic acid levels in the epithelium of both the lactic ducts and alveoli of lactic glands were examined in heifers, close to full term, lactating cows, some of them with subclinical mastitis lesions, and udder biopsies of two cows. The activities of SDH and LDH as well as RNA levels were pronounced in the glandular epithelium of alveoli and in the epithelium of the lesser lactic ducts. All three parameters were also pronounced but at lower levels in the cells of the apical stratum of the two-layer epithelium in the greater lactic ducts. Relationships were found to exist between the enzyme activities and RNA levels and the lactation cycle. Their patterns and manifestations were more or less typical of those areas of the lactic gland which were affected by mastitis.

Age Factors↗

PubMed1999

Role of mitochondrial lactate dehydrogenase and lactate oxidation in the intracellular lactate shuttle.

To evaluate the potential role of mitochondrial lactate dehydrogenase (LDH) in tissue lactate clearance and oxidation in vivo, isolated rat liver, cardiac, and skeletal muscle mitochondria were incubated with lactate, pyruvate, glutamate, and succinate. As well, alpha-cyano-4-hydroxycinnamate (CINN), a known monocarboxylate transport inhibitor, and oxamate, a known LDH inhibitor were used. Mitochondria readily oxidized pyruvate and lactate, with similar state 3 and 4 respiratory rates, respiratory control (state 3/state 4), and ADP/O ratios. With lactate or pyruvate as substrates, alpha-cyano-4-hydroxycinnamate blocked the respiratory response to added ADP, but the block was bypassed by addition of glutamate (complex I-linked) and succinate (complex II-linked) substrates. Oxamate increased pyruvate (approximately 10-40%), but blocked lactate oxidation. Gel electrophoresis and electron microscopy indicated LDH isoenzyme distribution patterns to display tissue specificity, but the LDH isoenzyme patterns in isolated mitochondria were distinct from those in surrounding cell compartments. In heart, LDH-1 (H4) was concentrated in mitochondria whereas LDH-5 (M4) was present in both mitochondria and surrounding cytosol and organelles. LDH-5 predominated in liver but was more abundant in mitochondria than elsewhere. Because lactate exceeds cytosolic pyruvate concentration by an order of magnitude, we conclude that lactate is the predominant monocarboxylate oxidized by mitochondria in vivo. Mammalian liver and striated muscle mitochondria can oxidize exogenous lactate because of an internal LDH pool that facilitates lactate oxidation.

Animals↗

PubMedDate not supplied

[Malate dehydrogenase and lactate dehydrogenase in trematodes and turbellarians].

Studies have been made on the activity and properties of malate and lactate dehydrogenases from the cattle rumen trematodes Eurytrema pancreaticum, Calicophoron ijimai and the turbellarian Phagocata sibirica which has a common free-living ancestor with the trematodes. All the species studied have a highly active malate dehydrogenase, its activity in the reaction of reducing oxaloacetate being 6-14 times higher than in the reaction of malate oxidation. The affinity of malate dehydrogenase to oxaloacetate was found to be higher than that to malate. The activity of lactate dehydrogenase (reducing the pyruvate) was lower than the activity of malate dehydrogenase, the difference being 50 times for C. ijimai, 4 times for E. pancreaticum and 10 times for P. sibirica.

Animals↗

PubMed1979

Double inhibition of D-glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase.

The simultaneous action of ATP (partially uncompetitive inhibitor with respect to Pi) and quinaldate (purely non-competitive inhibitor with respect to Pi) on D-glyceraldehyde-3-phosphate dehydrogenase was analyzed kinetically. The interaction constant [as defined by Keleti and Fajszi (1971) Math. Biosci. 12 197] of the two inhibitors for the D-glyceraldehyde-3-phosphate dehydrogenase-Pi complex is greater than 1, which means that the two inhibitors act antagonistically. The kinetic analysis of the double inhibition shows that there is no ATP-enzyme-quinaldate ternary complex, but a quaternary complex with Pi is formed. The interaction of the two inhibitors on the enzyme-Pi complex depends on substrate (Pi) concentration. The antagonistic effect of the two inhibitors becomes additive at low Pi concentrations (about 1 mM). The simultaneous action of oxalate (purely uncompetitive inhibitor with respect to NAD) and quinaldate (partially mixed type inhibitor with respect to NAD) on lactate dehydrogenase was also analyzed. Oxalate and quinaldate act antagonistically on lactate dehydrogenase. However, at low NAD concentrations (about 0.06 mM) or at high quinaldate and low oxalate concentrations (around 7 and 1.7 mM, respectively) the antagonism turns into the simple summation of the effects of the two inhibitors.

Adenosine Triphosphate↗

PubMed1997

Formation of homo- and heterooligomeric supramolecular structures by D-glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase in reversed micelles of aerosol OT in octane.

The supramolecular structure of oligomeric enzymes can be specifically regulated by changing the size of an inner cavity of Aerosol OT reversed micelles in octane. Both D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) reveal an ability to exist and function in monomeric, dimeric and tetrameric forms (homooligomers). Various heterooligomeric complexes, in particular, GAPDH monomer--LDH monomer, GAPDH dimer--LDH tetramer were detected in reversed micelles.

Animals↗

PubMed1982

Glycerol phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase: activities in oligodendrocytes, neurons, astrocytes, and myelin isolated from developing rat brains.

Glycerol phosphate dehydrogenase (GPDH), glucose-6-phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH) activities were determined in oligodendrocytes, neurons, and astrocytes isolated from the brains of developing rats. The activity of each enzyme was significantly lower in both neurons and astrocytes than in oligodendrocytes. The GPDH activity in oligodendrocytes increased more than 4-fold during development, and at 120 days cells of this type had 1.4-fold the specific activity of forebrain homogenates. The G6PDH activities in oligodendrocytes from 10-day-old rats were 1.4-fold the activities in the forebrain homogenates. The activities of this enzyme in oligodendrocytes were progressively lower at later ages, such that at 120 days the cells had 0.8 times the specific activities of homogenates. The oligodendrocytes had 0.6 times the homogenate activities of LDH at 10 days, and this ratio had decreased to 0.2 by 120 days. These enzymes were also measured in myelin isolated from 20-, 60-, and 120-day-old rats. By 120 days the specific activities of G6PDH and LDH in myelin were less than 8% of the respective activities in homogenates. The GPDH activity in myelin was, however, at least 20% the specific activity in the homogenates, even in the oldest animals. It is proposed that LDH could be used as a marker for oligodendroglial cytoplasm in subfractions of myelin and in myelin-related membrane vesicles.

Animals↗