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PubMed1989

Modulation of lactate dehydrogenase activity by enzyme-protein interaction.

Some lactate dehydrogenase modulator proteins have been isolated from the lactate dehydrogenase-free crude mitochondrial fraction of rabbit muscle, beef liver and chicken liver. It was shown that beef and chicken liver mitochondrial extracts exhibited activatory capacity in contrast to the inhibitory capacity of rabbit muscle mitochondrial extracts. All modulators can be precipitated by 80% ammonium sulphate saturation and show high anodic electrophoretic mobility and heat stability. Modulators have higher affinity for alkaline pI lactate dehydrogenase isoenzymes, independent of whether the M and H subunits are predominant. The inhibitor and the activator molecules compete for lactate dehydrogenase since their modulatory capacity was nullified when similar relative amounts were used. This study shows the existence of analogous proteins with an acidic pI in the different mitochondrial fractions which modify lactate dehydrogenase activity.

Animals↗
PubMed1986

Interaction of bovine heart lactate dehydrogenase with erythrocyte lipids.

The interaction between bovine heart lactate dehydrogenase and erythrocyte lipid suspension as a function of pH, NAD, NADH, lipid and salt concentration was studied by ultracentrifugation. In the presence of erythrocyte lipid liposomes the enzyme forms two kinds of complex: lactate dehydrogenase adsorbed to liposomes and soluble lactate dehydrogenase-phospholipid complexes. The two complexes reveal different dependence of their stability on pH values. Lactate dehydrogenase decreases its specific activity when it binds to the phospholipid molecules. Efficient adsorption of lactate dehydrogenase to liposomes occurs in their pH range 6.0-8.0 and at low ionic strength. The adsorption is diminished in the presence of NAD+ but it is not influenced by NADH. Possible mechanisms of the interaction and implications for the function in vivo are discussed.

Adsorption↗
PubMed1993

L-mandelate dehydrogenase from Rhodotorula graminis: comparisons with the L-lactate dehydrogenase (flavocytochrome b2) from Saccharomyces cerevisiae.

L-Lactate dehydrogenase (L-LDH) from Saccharomyces cerevisiae and L-mandelate dehydrogenase (L-MDH) from Rhodotorula graminis are both flavocytochromes b2. The kinetic properties of these enzymes have been compared using steady-state kinetic methods. The most striking difference between the two enzymes is found by comparing their substrate specificities. L-LDH and L-MDH have mutually exclusive primary substrates, i.e. the substrate for one enzyme is a potent competitive inhibitor for the other. Molecular-modelling studies on the known three-dimensional structure of S. cerevisiae L-LDH suggest that this enzyme is unable to catalyse the oxidation of L-mandelate because productive binding is impeded by steric interference, particularly between the side chain of Leu-230 and the phenyl ring of mandelate. Another major difference between L-LDH and L-MDH lies in the rate-determining step. For S. cerevisiae L-LDH, the major rate-determining step is proton abstraction at C-2 of lactate, as previously shown by the 2H kinetic-isotope effect. However, in R. graminis L-MDH the kinetic-isotope effect seen with DL-[2-2H]mandelate is only 1.1 +/- 0.1, clearly showing that proton abstraction at C-2 of mandelate is not rate-limiting. The fact that the rate-determining step is different indicates that the transition states in each of these enzymes must also be different.

Alcohol Oxidoreductases↗
PubMedDate not supplied

[Effect of freezing-thawing on the activity of lactate dehydrogenase isoenzymes].

The activity of isolated M4- and H4-lactate dehydrogenase isoenzymes is studied after freezing in the saline solution to -8, -9.5, -23 and -30 degrees C. It is shown that the activity of H4-lactate dehydrogenase does not change, M4-lactate dehydrogenase being partially inactivated. It is demonstrated that a reduction in the M4-LDH activity is due to the effect of high salt concentrations on the freezing-out water from the solution and is an irreversible process. The fluorescence spectra of these isoenzymes are studied after freezing-thawing, and the interpretation of the observed changes in these spectra is suggested on the basis of possible alterations in the quaternary structure and sonformation of the enzyme molecule subunits. It is shown that the substrates and coenzymes of lactate dehydrogenase (NAD, NADH, pyruvate, lactate) completely or partially prevent the inactivation of M4-isoenzymes at freezing-thawing.

Animals↗
PubMed1983

A fructose bisphosphate activated lactate dehydrogenase in the liver fluke Fasciola hepatica.

Lactate dehydrogenase of Fasciola hepatica showed typical Michaelis-Menten kinetics at pH 7.2, with respect to pyruvate. Addition of physiological levels of fructose bisphosphate activated the enzyme at all substrate concentrations tested; the response to this effector being hyperbolic in nature. As well as depending upon the fructose bisphosphate concentration, the Vmax and Km are modified by different buffers. The degree of activation is much greater using Tris-HCl than phosphate buffer. The pH optimum occurs at pH 6.5 whether using physiological levels of substrate in the presence or absence of fructose bisphosphate, or high levels of substrate. Of the potential effectors tested, significant inhibition was shown by the nucleoside triphosphates, especially ATP. The importance of this inhibition, coupled with the activation by fructose bisphosphate is discussed. Fasciola hepatica lactate dehydrogenase is unusual in that it does not catalyse the reverse reaction to any measurable extent. That is, lactate oxidation is negligible unless the effector fructose bisphosphate is present. Use was made of this fact to visualise the isoenzymes of lactate dehydrogenase separated by polyacrylamide disc gel electrophoresis. Five isoenzyme bands became apparent when stained in this manner.

Animals↗
PubMedDate not supplied

[Properties of lactate dehydrogenase of brain mitochondria].

The activity of lactate dehydrogenase in the brain mitochondria isolated within the phycol density gradient accounts for 5% of the enzyme activity determined in cytoplasm. More than 80% of this activity is found in the intermembrane space of mitochondria. Purification, washing and incubation of the mitochondria with supernatant fluid do not change the activity. The treatment with lubrol, desoxycholate or ultrasound evoked almost a two-fold increase in the enzymic activity in the mitochondria and does not change it in the supernatant fluid. Rotenone and Ca2+ have no effect on the activity of lactate dehydrogenase in the both preparations, KCN inhibits it. Mg2+ increases the activity in the supernatant fluid and decreases it in the mitochondria. The relative amount of M- and N- forms of lactate dehydrogenase is approximately the same in the both studied objects.

Animals↗
PubMed1980

Modification of mouse testicular lactate dehydrogenase by pyridoxal 5'-phosphate.

1. Mouse C4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.7 and 25 degrees C loses activity gradually; 1mM-pyridoxal 5'-phosphate causes 83% inactivation, and higher concentrations of the reagent cause no further loss of activity. 2. The final extent of inactivation is very pH-dependent, greater inactivation occurring at the high pH values. 3. Inactivation may be fully reversed by addition of cysteine, or made permanent by reducing the enzyme with NaBH4. 4. The absorption spectrum of inactivated reduced enzyme indicates modification of lysine residues. Inactivation by 80% corresponds to modification of at least 1.8 mol of lysine/mol of enzyme subunit. 5. There is no loss of free thiol groups after inactivation with pyridoxal 5'-phosphate and reduction of the enzyme. 6. NAD+ or NADH gives complete protection against inactivation. protection studies with coenzyme fragments indicate that the AMP moiety is largely responsible for the protective effect. Lactate (10 mM) gives no protection in the absence of added nucleotides, but greatly enhances the protection given by ADP-ribose (1 mM). Thus ADP-ribose is able to trigger the binding of lactate. 7. Pyridoxal 5'-phosphate also acts as a non-covalent inhibitor of mouse C4 lactate dehydrogenase. The inhibition is non-competitive with respect to both NAD+ and lactate. 8. Km values for the enzyme at pH 8.0 and 25 degrees C, with the non-varied substrate saturating, are 0.3 mM-lactate and 5 microM-NAD+. 9. These results are discussed and compared with pyridoxal 5'-phosphate modification of other lactate dehydrogenase isoenzymes and related dehydrogenases.

Animals↗
PubMedDate not supplied

Distribution of lactate dehydrogenase isoenzymes in coelomic fluid and fetal adnexae.

The objective of this study was to evaluate the distribution of lactate dehydrogenase isoenzymes in the human gestational sac. Total lactate dehydrogenase and its isoenzymes were measured in matched coelomic fluid and samples of villous and decidual tissues collected at the time of pregnancy termination in a group of 16 healthy women who were between 7 and 12 weeks of gestation. In addition intact secondary yolk sacs (n = 2) and fragments of fetal liver (n = 3) were collected. For comparison, samples of placental tissue, amniotic membrane and chorion obtained at term, were also investigated. In early pregnancy, H-type lactate dehydrogenase isoenzymes predominate in placental villous tissue and coelomic fluid, whereas M-type lactate dehydrogenase isoenzymes predominate in extracts of decidua, secondary yolk sac and fetal liver. These results highlight the important contribution of placental bioproducts to the constitution of the coelomic fluid. We observed also that placental patterns of lactate dehydrogenase isoenzymes change between the first and the third trimester of gestation, from the H-type to the M-type lactate dehydrogenase isoenzymes. We suggest that the lactate dehydrogenase pattern might be an indicator of proliferative or differentiative potential of the trophoblastic cells throughout pregnancy.

Adnexa Uteri↗
PubMed1995

Plasmodium falciparum and Plasmodium vivax: lactate dehydrogenase activity and its application for in vitro drug susceptibility assay.

Lactate dehydrogenase, the terminal enzyme of anerobic Embden-Meyerhoff glycolysis, plays an important role in the carbohydrate metabolism of human malaria parasites. Based on the ability of malarial lactate dehydrogenase to use 3-acetylpyridine NAD as a coenzyme in a reaction leading to the formation of pyruvate from L-lactate, the enzymatic activity of fresh clinical isolates of Plasmodium falciparum and Plasmodium vivax was determined in relation to incubation time, asexual stages, and parasitemia and applied to a drug susceptibility assay. Lactate dehydrogenase activity was detectable at a parasitemia > 0.4%, at a hematocrit of 1.5%, and increased with parasitemia. Maximal lactate dehydrogenase activity was generally observed between 36 and 48 hr, when the trophozoites and schizonts predominated. The results of the in vitro drug susceptibility assays based on the inhibition of lactate dehydrogenase activity and on the incorporation of tritium-labeled hypoxanthine were correlated. For an optimal performance against fresh clinical malaria isolates, however, the enzymatic assay requires an initial parasitemia between 1 and 2% at a hematocrit of 1.5%.

Animals↗
PubMed1994

Diagnostic and prognostic significance of lactate dehydrogenase in cerebrospinal fluid in patients of meningitis.

Activity of lactate dehydrogenase was measured in cerebrospinal fluid in 60 cases of meningitis (35 pyogenic, 18 tuberculous and 7 viral). Thirty age and sex matched healthy controls were taken to find out its diagnostic and prognostic significance in meningitis. The cerebrospinal fluid lactate dehydrogenase activity was significantly higher (p < 0.001) in cases of pyogenic meningitis and tuberculous meningitis as compared to the control group. The maximum elevation was seen on the 1st day in all types of meningitis but the activity declined significantly thereafter in patients of pyogenic meningitis who recovered without complications. However, in 2 cases of pyogenic meningitis and 5 cases of tuberculous meningitis who survived with complications, the lactate dehydrogenase activity on subsequent estimations increased serially. Further, the basal lactate dehydrogenase activity in 9 cases of pyogenic meningitis (who expired) was higher than those who survived without complications. The basal lactate dehydrogenase activity in pyogenic meningitis was significantly higher (p < 0.05) as compared to tuberculous meningitis and viral meningitis patients (p < 0.02) in this study.

Adolescent↗
PubMed1992

Glutamate 264 modulates the pH dependence of the NAD(+)-dependent D-lactate dehydrogenase.

Recently, we amplified the Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene by the polymerase chain reaction, cloned and overexpressed it in Escherichia coli (Kochhar, S., Chuard, N., and Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 185, 705-712). Polymerase chain reaction-amplified DNA fragments may contain base changes resulting in mutant gene products. A comparison of specific activities of D-lactate dehydrogenase in the crude extracts of 50 recombinant clones indicated that one of the clones had drastically reduced enzyme activity. Nucleotide sequence analysis of the insert DNA showed an exchange of A to G at position 795 resulting in substitution of Glu264 to Gly in the D-lactate dehydrogenase. The purified mutant D-lactate dehydrogenase showed a shift of 2 units in its optimum pH toward the acidic range. The dependence of kcat/Km on the pH of the mutant enzyme showed that the pKa of the free enzyme was around 4, at least 2 pH units lower than that of the wild-type enzyme. Both the wild-type and the mutant enzyme at their respective optimum pH values showed similar kcat and Km values. The data suggest that the highly conserved Glu264 is not critical for enzyme catalysis, but it must be situated within hydrogen bonding distance to amino acid residue(s) involved in substrate binding as well as in catalysis.

Amino Acid Sequence↗
PubMed1976

A comparison of some kinetic properties of soluble and bound lactate dehydrogenase isoenzymes at different temperatures.

A comparison was made of some kinetic properties of three chicken lactate dehydrogenase isoenzymes (1, 3 and 5) at 4, 16, 23 and 40 degrees C. Assays were performed with an enzyme concentration of 0.01 muM at pH 6.0. Under the conditions of assay, lactate dehydrogenase 3 and 5 bound to the particulate fraction of homogenized skeletal muscle and were evaluated in the soluble and particulate state. Binding of isoenzymes 3 and5 to the cellular particulate fraction decreased V. This decrease was much greater for lactate dehydrogenase 5 and 3. Values of V for lactate dehydrogenase isoenzymes 1 and 3 did not follow a simple Arrhenius relationship; there was a rapid change in activity between 16 and 23 degrees C. The apparent Km (pyruvate) values of all isoenzymes (bound or soluble) increased with increasing temperature, changing 4--10-fold. The apparent Km for lactate dehydrogenase 5 was greater than that for lactate dehydrogenase 3, which in turn was greater than that for lactate dehydrogenase 1.

Animals↗
PubMed1985

Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Location and regulation of expression.

Acinetobacter calcoaceticus possesses an L(+)-lactate dehydrogenase and a D(-)-lactate dehydrogenase. Results of experiments in which enzyme activities were measured after growth of bacteria in different media indicated that the two enzymes were co-ordinately induced by either enantiomer of lactate but not by pyruvate, and repressed by succinate or L-glutamate. The two lactate dehydrogenases have very similar properties to L(+)-mandelate dehydrogenase and D(-)-mandelate dehydrogenase. All four enzymes are NAD(P)-independent and were found to be integral components of the cytoplasmic membrane. The enzymes could be solubilized in active form by detergents; Triton X-100 or Lubrol PX were particularly effective D(-)-Lactate dehydrogenase and D(-)-mandelate dehydrogenase could be selectively solubilized by the ionic detergents cholate, deoxycholate and sodium dodecyl sulphate.

Acinetobacter↗
PubMed1975

Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles.

In the preceding paper the preparation and characterization of antiserum to purified D-lactate are described. In this paper the effects of the antibody on D-lactate dehydrogenase activity and D-lactate-dependent active transport in native Escherichia coli ML 308-225 membrane vesicles and ML 308-225dld-3 vesicles reconstituted with D-lactate dehydrogenase are described. The results demonstrate that D-lactate dehydrogenase is inaccessible to antibody in native ML 308-225 vesicles, but readily accessible to antibody in reconstituted dld-3 vesicles. The findings indicate that D-lactate dehydrogenase is located on the inner surface of native ML 308-225 vesicles and on the outer surface of reconstituted dld-3 vesicles. The results with the native vesicle preparations also provide further evidence that virtually none of the vesicles is inverted or sufficiently damaged to allow access of antibody to D-lactate dehydrogenase. In addition, experiments are presented which demonstrate that an impermeable electron carrier, reduced 5-N-methylphenazonium-3-sulfonate, drives active transport in native ML 308-225 vesicles as well as its permeable analogue reduced phenazine methosulfate. Thus, reduction of the respiratory chain from either side of the vesicle membrane is able to drive active transport. Ca2+, Mg2+-stimulated ATPase is also inaccessible to antibody in ML 308-225 vesicles unless the preparation is subjected to ultrasonic sound, incubated in Tris buffer at pH 9.0, or homogenized vigorously. Moreover, as opposed to D-lactate dehydrogenase and cytochrome b1, ATPase is readily lost from the membrane during the preparation of vesicles.

Animals↗
PubMed1975

Purification and properties of a fructose-1,6-diphosphate activated L-lactate dehydrogenase from Staphylococcus epidermidis.

L-(+)-lactate dehydrogenase (LDH) from Staphylococcus epidermidis ATCC 14990 was purified by affinity chromatography. The purified enzyme was specifically activated by fructose-1,6-diphosphate (FDP). The concentration of FDP required for 50% maximal activity was about 0.15 mM. The enzyme activity was inhibited by adenosine diphosphate (ADP) and oxamate. The inhibition by ADP appeared to be competitive with respect to reduced nicotinamide adenine dinucleotide (NADH). The catalytic activity of the LDH for pyruvate reduction exhibited an optimum at pH 5.6. The enzyme is composed of four, probably identical, subunits. Sephadex gel filtration and sedimentation velocity at pH 5.6 Yielded molecular weights of about 130 000 and 126 000, respectively. The molecular weight at pH 6.5 and 7.0 was found to be only about 68 000. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and sedimentation velocity at pH 2.0 or 8.5 revealed monomeric subunits with an approximate molecular weight of 36000. The thermostability of the heat labile enzyme was increased in the presence of FDP, NADH and pyruvate. The purified LDH exhibited an anomalous type of kinetic behavior. Plots of initial velocity vs. different concentrations of pyruvate, NADH or FDP led to saturation curves with intermediary plateau regions. As a consequence of these plateau regions the Hill coefficient alternated between lower and higher n-values. Some distinguishing properties of the S. epidermidis LDH and other LDHs activated by FDP are discussed.

Adenine Nucleotides↗
PubMedDate not supplied

Mouse Ia antigens are receptors for lactate dehydrogenase virus.

Infection of mice with lactate dehydrogenase virus (LDV) leads to elevation of plasma lactate dehydrogenase, lifelong viraemia and perturbations of cell-mediated and humoral immune responses. The virus replicates exclusively in a restricted set of macrophages, but the basis for restricted cell susceptibility is unknown. By immunofluorescence techniques we have found that the per cent infected was the same as the per cent expressing antigens encoded by the I region of the major histocompatibility complex (Ia). Infection of CBA strain I-A+ peritoneal macrophages was blocked when cells were treated simultaneously with monoclonal antibody to I-A and I-E, but not with either antibody separately. LDV infectivity was inactivated when virus was treated with purified rat glycoprotein homologous to mouse I-A and I-E antigens. These results indicate that the receptors for LDV are I-A and I-E antigens. Selective infection of Ia-positive macrophages may have an important effect on the immunological capability of infected mice.

Animals↗
PubMed1975

Serum enzyme activity of hydroxybutyric dehydrogenase (HBD) and heat inactivated lactate dehydrogenase (LD) after operations on intracranial aneurysms.

The relation between clinical signs of parenchymatous cell damage after brain surgery and the serum activity of the LD-isoenzymes determined either as HBD or as heat inactivated LD, has been studied in 44 patients operated upon for intracranial aneurysms. Seven patients had a postoperative increase of the serum enzyme activity above the upper normal limit. All these showed neurological symptoms as compared to only 14% of those who had a postoperative enzyme level within normal limits. This speaks strongly in favour of a relation between the brain cell damage and the increase of the serum activity of the LD-isoenzyme. Since only 15% of the patients had a pathological enzyme level, it is further suggested that an uncomplicated operation for intracranial aneurysm should not cause an increase of the HBD or heat inactivated LD above the upper normal limit.

Adult↗