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PubMed1988

Acute and chronic effects of insulin on lactate dehydrogenase (LDH) activity in tissues of albino rat (Wistar strain).

Male albino rats were treated with insulin for one week (acute) and four weeks (chronic). The lactate dehydrogenase (LDH) activity, lactate and pyruvate levels were estimated in the tissues of experimental and control animals. LDH activity decreased in all the tissues of acute- and chronic treated animals whereas the lactate content is elevated. Pyruvate content also showed increment except in heart and pancreas with reference to acute treatment where it is decreased. The hyperinsulinaemia effect in relation to lacticacidaemia and its influence on energy demand and ammonia secretion is discussed.

Animals↗

PubMed1989

On the effect on specificity of Thr246----Gly mutation in L-lactate dehydrogenase of Bacillus sterothermophilus.

The function of the amino acid Thr246 in L-lactate dehydrogenase from Bacillus stearothermophilus has been investigated by site-directed replacement with glycine. Kinetic experiments with a number of 2-oxo acids showed strongly reduced activity for the mutated enzyme. However, the mutant enzyme shows a relative preference for the large hydrophobic sidechains of alpha-keto acids and an even higher specific activity than the wild-type lactate dehydrogenase for the polar oxaloacetate substrate. Graphic analyses indicate that the loss of one hydrogen bond, or intrusion of water into the active site, might be responsible for the reduced activity. The kinetic results suggest that the binding modes of bulky hydrophobic or polar substrates compensate to some degree for the partially disrupted active site.

Amino Acid Sequence↗

PubMed1979

The tentative amino acid sequencing of lactate dehydrogenase C4 by X-ray diffraction analysis.

A tentative amino acid sequence of mouse testicular lactate dehydrogenase C4 was deduced from an electron density map and comparison with five other known lactate dehydrogenase sequences. The amino acid composition determined by chemical analysis agrees reasonably well with the present results. Necessary changes in amino acids were largely conservative and confined to the external portions of the molecule. Residues in the Q and P subunit contact regions were particularly well conserved as were most internal residues. The minimum base change/codon was similar between the C and H isoenzymes and between the C and M isoenzymes.

Amino Acid Sequence↗

PubMed1975

Modification of pig M4 lactate dehydrogenase by pyridoxal 5'-phosphate. Demonstration of an essential lysine residue.

1. Pig M4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.5 and 25 degrees C loses activity gradually. The maximum inactivation was 66%, and this did not increase with concentrations of pyridoxal 5'-phosphate above 1 mM. 2. Inactivation may be reversed by dialysis or made permanent by reducing the enzyme with NaBH4. 3. Spectral evidence indicates modification of lysine residues, and 6-N-pyridoxyl-lysine is present in the hydrolsate of inactivated, reduced enzyme. 4. A second cycle of treatment with pyridoxal 5'-phosphate and NaBH4 further decreases activity. After three cycles only 9% of the original activity remains. 5. Apparent Km values for lactate and NAD+ are unaltered in the partially inactivated enzyme. 6. These results suggest that the covalently modified enzyme is inactive; failure to achieve complete inactivation in a single treatment is due to the reversibility of Schiff-base formation and to the consequent presence of active non-covalently bonded enzyme-modifier complex in the equilibrium mixture. 7. Although several lysine residues per subunit are modified, only one appears to be essential for activity: pyruvate and NAD+ together (both 5mM) completely protect against inactivation, and there is a one-to-one relationship between enzyme protection and decreased lysine modification. 8. NAD+ or NADH alone gives only partial protection. Substrates give virtually none. 9. Pig H4 lactate dehydrogenase is also inactivated by pyridoxal 5'-phosphate. 10. The possible role of the essential lysine residue is discussed.

Animals↗

PubMed1987

Adaptative features of enzymes from family Sciaenidae--III. Studies on lactate dehydrogenase (LDH) of fishes from the south coast of Uruguay.

1. Electrophoretic analyses of lactate dehydrogenase isozyme patterns of three species of temperate fish belonging to family Sciaenidae (order Perciformes) indicates that at least three LDH loci--Ldh-A, Ldh-B and Ldh-C are active. 2. The subunits encoded by these loci occurred at different levels in the different tissues and organs analyzed. 3. The products of these loci show different response to changes in temperature. The results were obtained by electrophoretic analyses. 4. Thermostability of skeletal muscle, heart and eye lactate dehydrogenase incubated at 60 degrees C for different periods of time were examined. 5. Relative activities of LDH isozymes were compared by Klebe's method to determine pattern of divergence of duplicated gene expression in the three species studied.

Acclimatization↗

PubMed1982

Effect of guanidination on subunit interactions in hybrid isozymes from pig lactate dehydrogenase.

The thermostability of the isozymes from pig heart (H) and muscle (M) lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) decreased in the order of H4 greater than M4 greater than H3M greater than HM3 greater than H2M2, while the thermostability of the isozymes from guanidinated H4 and M4 increased as guanidinated H monomer was substituted by M monomer. The increased thermostability of H4 increased as guanidinated H monomer was substituted by M monomer. The increased thermostability of H4 on guanidination of five lysine residues per subunit was due to the decrease in the standard activation entropy, and no change in the standard activation enthalpy was observed. The more increased thermostability of H4 on further guanidination of lysine residues from 5 to 15 per subunit was due to the increase of the standard activation enthalpy which overcame the decrease in thermostability due to the increase of the standard activation entropy. The results indicate two different mechanisms of stabilization depending on the degree of guanidination. The increase of thermostability, as measured by the change of the standard activation free energy for thermal inactivation of H2M2, was almost the same as that of H4 on guanidination of five lysine residues in an H monomer. This result and the order of thermostability of the isozymes from unmodified and guanidinated H4 and M4 suggest that the increase of thermostability of hybrid isozymes on guanidination of H monomer is due to the change of the heterologous subunit interactions.

Animals↗

PubMed2001

The Membrane-bound L- and D-lactate dehydrogenase activities in mitochondria from Euglena gracilis.

The activity of the pyridine nucleotide-independent lactate dehydrogenase (iLDH) was characterized in mitochondria isolated from the protist Euglena gracilis. The dissociation constants for L- and D-lactate were similar, but the V(max) was higher with the d isomer. A ping-pong kinetic mechanism was displayed with 2,4-dichlorophenol-indolphenol (DCPIP), or coenzyme Q(1), reacting as the second substrate with the modified, reduced enzyme. Oxamate was a competitive inhibitor against both L- and D-lactate. Oxalate exerted a mixed-type inhibition regarding L- or D-lactate and also against DCPIP. The rate of L-lactate uptake was partially inhibited by mersalyl and lower than the rate of dehydrogenation, which was mersalyl-insensitive. These data suggested that the active site of L-iLDH was orientated toward the intermembrane space. The following observations indicated the existence of two stereo-specific iLDH enzymes in the inner membrane of Euglena mitochondria: a greater affinity of the D-iLDH for both inhibitors, D-iLDH thermo-stability at 70 degrees C and denaturation of L-iLDH, opposite signs in the enthalpy change for the association reaction of the isomers to the enzyme, differential solubilization of both activities with detergents, and different molecular mass.

Animals↗

PubMed1991

Kinetic mechanism of the endogenous lactate dehydrogenase activity of duck epsilon-crystallin.

Initial velocity, product inhibition, and substrate inhibition studies suggest that the endogenous lactate dehydrogenase activity of duck epsilon-crystallin follows an order Bi-Bi sequential mechanism. In the forward reaction (pyruvate reduction), substrate inhibition by pyruvate was uncompetitive with inhibition constant of 6.7 +/- 1.7 mM. In the reverse reaction (lactate oxidation), substrate inhibition by L-lactate was uncompetitive with inhibition constant of 158 +/- 25 mM. The cause of these inhibitions may be due to epsilon-crystallin-NAD(+)-pyruvate and epsilon-crystallin-NADH-L-lactate abortive ternary complex formation as suggested by the multiple inhibition studies. Pyruvate binds to free enzyme very poorly, with a very large dissociation constant. Bromopyruvate, fluoropyruvate, pyruvate methyl ester, and pyruvate ethyl ester are alternative substrates for pyruvate. 3-Acetylpyridine adenine dinucleotide, nicotinamide 1,N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide serve as alternative coenzymes for epsilon-crystallin. All the above alternative substrates or coenzymes showed an intersecting initial-velocity pattern conforming to the order Bi--Bi kinetic mechanism. Nicotinic acid adenine dinucleotide, thionicotinamide adenine dinucleotide, and 3-aminopyridine adenine dinucleotide acted as inhibitors for this enzymatic crystallin. The inhibitors were competitive versus NAD+ and noncompetitive versus L-lactate. alpha-NAD+ was a noncompetitive inhibitor with respect to the usual beta-NAD+. D-Lactate, tartronate, and oxamate were strong dead-end inhibitors for the lactate dehydrogenase activity of epsilon-crystallin. Both D-lactate and tartronate were competitive inhibitors versus L-lactate while oxamate was a competitive inhibitor versus pyruvate. We conclude that the structural requirements for the substrate and coenzyme of epsilon-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity.

Animals↗

PubMed1983

Identification of two distinct lactate dehydrogenases in Rhodospirillum rubrum.

The activities of pyridine nucleotide-independent d- and l-lactate dehydrogenases were detected in membranes from Rhodospirillum rubrum grown under aerobic and phototrophic conditions. Crossed immunoelectrophoretic analysis revealed two antigenically distinct enzymes that were further distinguished by specificity for d- and l-stereoisomers of lactate and by the sensitivity of the d-lactate dehydrogenase to inhibition by oxamate and oxalate.

Immunoelectrophoresis, Two-Dimensional↗

PubMedDate not supplied

[Changes in lactate dehydrogenase activity in chicken tissues in hyperthyreosis in ontogenesis].

The lactate dehydrogenase activity in reactions of lactate oxidation and synthesis was studied in subfractions of the chicken brain, heart and liver at the embryonal, early postembryonal and adult stages of development after thyroxine administration. It has been shown that during embryogenesis thyroxine predominantly enhanced the rate of lactate oxidation in the mitochondrial tissues. A marked increase in the lactate synthesis was found in cytoplasm of the adult chicken tissues. Specificity of enzyme activity alterations was detected in the chicken brain during ontogenesis after thyroxine administration.

Animals↗

PubMed1975

Pig heart lactate dehydrogenase. Binding of pyruvate and the interconversion of pyruvate-containing ternary complexes.

1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2. In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results.

Binding Sites↗

PubMed1990

Cerebrospinal fluid lactate dehydrogenase levels in early stroke and transient ischemic attacks.

We examined the concentrations of lactate dehydrogenase in the cerebrospinal fluid of 25 patients with strokes and 15 patients with transient ischemic attacks less than or equal to 8 hours after the onset of the vascular event and in a control group of 21 patients. We found significantly higher concentrations in the stroke patients (40.9 +/- 14.5 units/l) than in the transient ischemic attack patients (11.8 +/- 2.9 units/l, p less than 0.001) and the controls (11.2 +/- 6.7 units/l, p less than 0.001). Among the stroke patients, we found a significantly higher lactate dehydrogenase concentration in those with cortical strokes (n = 12, 50 +/- 12.3 units/l) than in those with lacunar white matter infarcts (n = 5, 26.4 +/- 6.5 units/l; p less than 0.001) and those with basal ganglia infarcts (n = 8, 36.37 +/- 11.7 units/l; p less than 0.05). Our study offers a supplementary examination for diagnosing cortical or subcortical infarction during the early stage of the event, with the possibility of distinguishing precisely stroke from transient ischemic attack during the first hours after onset of the event.

Acute Disease↗

PubMed1989

Increase of creatine kinase and lactate dehydrogenase in the serum of rats submitted to experimental intestinal infarction.

Acute intestinal ischemia is a severe condition for which no early biochemical index exists at present. We have investigated the effect of experimental intestinal infarction in the rat on the serum concentration of total creatine kinase, creatine kinase BB and total lactate dehydrogenase. Suppression of blood supply from the superior mesenteric artery resulted in marked increases of total creatine kinase, of its BB isoenzyme, and of total lactate dehydrogenase. Whereas total creatine kinase and total lactate dehydrogenase cannot be regarded as specific markers of the intestine, creatine kinase BB could prove to be more specific in view of its limited tissue distribution.

Animals↗

PubMed1994

A continuous spectrophotometric assay for ureidoglycolase activity with lactate dehydrogenase or glyoxylate reductase as coupling enzyme.

A spectrophotometric assay for ureidoglycolase activity (both ureidoglycolate lyase and hydrolase), based on the reduction of glyoxylate to glycolate catalyzed by glyoxylate reductase or lactate dehydrogenase with the stoichiometric and continuous NADH oxidation, is described. The assay has been optimized for the amount of coupling enzyme, reagent concentrations, buffers, and the nonenzymatic degradation of ureidoglycolate. Under optimal assay conditions, ureidoglycolase activity can be followed with either lactate dehydrogenase or glyoxylate reductase as coupling enzyme and reaction can be started by addition of substrate or enzyme extract. Once the reaction was started, NADH oxidation was linear with time after a lag phase of 1-2 min. This linear NADH oxidation was directly proportional to enzyme concentration in the assay mixture until changes in absorbance of 0.12 per minute. This method is easy and reliable for the accurate determination of ureidoglycolase activity in crude and purified extracts from Chlamydomonas reinhardtii cells and no notable interferences have been detected. Since lactate dehydrogenase is much cheaper and has a lower Km for its substrate than glyoxylate reductase and can be used as supplied, its use as the coupling enzyme of choice is recommended.

Alcohol Oxidoreductases↗

PubMed1986

Localization of L-lactate dehydrogenase in mitochondria.

Relatively small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from liver of the rat. Using a variety of cytosolic markers, it was found that essentially no cytosolic contamination was present. Respiratory velocities and respiratory control with L-lactate were somewhat lower than with glutamate, but equal or superior to those with pyruvate. Agarose gel electrophoresis showed LDH isoenzymes in mitochondria similar to that in corresponding cytosol. Subtilisin BPN', a bacterial protease, was incubated with intact mitochondria and enzyme activities were measured. Following mitochondrial disruption, the proteolytic treatment was repeated. Digitonin was also used in the fractionation of mitochondria. These techniques helped to determine the location of the LDH in the mitochondria as being mainly in the outer membrane and periplasmic space.

Animals↗

PubMed1994

[Lactate dehydrogenase isoenzymes in mammalian and chicken serum].

In the article we describe characteristics of lactate dehydrogenase (LD) (EC 1.1.1.27) isoenzymes of fowl origin. We compare them with characteristics of evolutionary different mammalian forms of the enzyme. Separations of LD isoenzymes have been done using the most progressive electrophoretic techniques available at present. They included electrophoresis in homogeneous and gradient polyacrylamide gels (PAGE) as well as isoelectric focusing (IEF). A typical densitometric pattern of serum LD isoenzymes obtained by gradient PAGE is illustrated in Fig. 1. It is obvious that LD isoenzymes of all investigated animals have been well separated under applied experimental conditions, with an exception of fowl serum. We succeeded in separating fowl LD isoenzymes using isoelectric focusing. It was possible to distinguish five fractions of lactate dehydrogenase (Fig. 2) by this technique. As shown in Fig. 2, mobility of avian isoenzymes in the gradient of pH differs from that of their mammalian analogues. Fowl LD isoenzymes were localized in cathodic part of IEF-gram. In the case of mammalian isoenzymes (cattle and rabbit), we found LD4 and LD5 forms of the enzyme in this part. The major fractions, i.e. LD1 to LD3 were present in anodic part of IEF-gram. We quantitatively expressed this different mobility of mammalian and avian forms of LD using retention factors Rf (Tab. III). A migration distance of cattle LD1 served as a reference point. It could be seen from a comparison of Rf values that avian forms of LD (of galiform origin) differed from mammalian ones by an outstanding shift of their isoelectric points, especially that of LD1, toward basic values. The total LD activity (IU) and the relative distribution of the LD isoenzyme activities (%) in normal serum of various animals are listed in Tabs I and II. A comparison of these values showed that, in contrast to mammalian serum (with the exception of the rat one), LD5 was the predominant fraction in fowl serum, followed by LD4. LD1 to LD3 occurred in low amounts with fairly similar proportions. On the other hand, most of mammalian serum revealed a reverse pattern of LD isoenzymes, i.e. predominant portion of serum activity had been concentrated in the first three anodic fractions and LD4 and LD5 had been found to be minor ones.

Animals↗

PubMed1973

Esterase, malate, and lactate dehydrogenases activity in murine neuroblastoma.

Mouse neuroblastoma tumors have only the fifth isozyme band (A(4)) of lactate dehydrogenase, whereas this band is missing in the brain which contained four other bands of lactate dehydrogenase. The alpha-esterase isozyme patterns of tumors, kidney, and brain are similar except that there is an additional slowest-moving form of esterase in all tumor tissues. The malate dehydrogenase pattern is not altered in any of the tissues.

Animals↗

PubMed1992

Effects of proteolytic and thermal action on the activity of lactate dehydrogenase isozymes LD-1 and LD-5.

L-Lactate dehydrogenase (LD) catalyzes the interconversion of pyruvate and lactate. Using a spectrophotometric assay to determine LD activity, incubation of rabbit, porcine, and bovine LD-1 and LD-5 isozymes with the protease subtilisin (Carlsberg) gave first-order degradation kinetics. Degradation half-lives were significantly lower for the LD-5 isozymes from the three species when incubated with subtilisin at temperatures from 4 degrees C to 25 degrees C. The energy involved in the degradation process, however, was not different. The activation energy for the conversion of pyruvate to lactate by LD-1 at pH 7.4 was significantly higher than that for LD-5 for all three species examined (P less than 0.005). Thermocalorimetry showed that the LD-1 isozymes have both a higher mean temperature of denaturation and a higher heat uptake during the denaturation process than corresponding LD-5 forms. The results suggest that the LD-5 isozymes in the species studied are more metabolically efficient, whereas the LD-1 forms have greater structural stability.

Animals↗