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PubMed1973

The kinetics of the interconversion of intermediates of the reaction of pig muscle lactate dehydrogenase with oxidized nicotinamide-adenine dinucleotide and lactate.

Oxamate competes with pyruvate for the substrate binding site on the E(NADH) complex of pig skeletal muscle lactate dehydrogenase. When this enzyme was mixed with saturating concentrations of NAD(+) and lactate in a stopped-flow rapid-reaction spectrophotometer there was no transient accumulation of enzyme complexes with the reduced nucleotide. The steady-state rate of formation of free NADH was reached within the dead-time of the instrument (3ms). When oxamate was added to inhibit the steady state and to uncouple the equilibration: [Formula: see text] through the rapid formation of E(NADH) (Oxamate), the rate of formation of E(NADH) could be measured by observation of the first turnover. This pH-dependent transient is controlled by the rate of dissociation of pyruvate and the fraction of the enzyme in the form E(NADH) (Pyruvate).

Animals↗

PubMed1967

Some properties of lactate dehydrogenase found in human urine.

1. Urinary lactate dehydrogenase was concentrated and subjected to starchblock electrophoresis. The isoenzyme pattern obtained was shown to be similar to that of the kidney cortex and medulla but different from that of the bladder and kidney pelvis. 2. The values for the relative activity and Michaelis constant of urinary lactate dehydrogenase were similar to those for kidney cortex and medulla but significantly different from those obtained for bladder and kidney pelvis. 3. The molecular weight of urinary lactate dehydrogenase was estimated by thin-layer chromatogtaphy on Sephadex G-200. The values obtained for several samples of urine ranged from 129 000 to 155 000 and were very close to that of the crystalline rabbit-muscle enzyme (140 000). 4. The question of the possible origin of urinary lactate dehydrogenase is discussed and the conclusion drawn that the kidney and not the plasma is the most likely source.

Chemical Phenomena↗

PubMed1991

Characterization of the fructose 1,6-bisphosphate-activated, L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus.

The L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus wt was purified to a final specific activity of 598 mumol pyruvate reduced per min per mg of protein. The specific activity of the pure enzyme with L(+)-lactate was 0.79 units per mg of protein. The M(r) of the native enzyme was 134,000 containing a single subunit type of M(r) 33,500 indicating an apparent tetrameric structure. The L(+)-lactate dehydrogenase was activated by fructose 1,6-bisphosphate in a cooperative manner affecting Vmax and Km values. The activity of the enzyme was also effected by pH, pyruvate and NADH. The Km for NADH at pH 6.0 was 0.05 mM and the Vmax for pyruvate reduction at pH 6.0 was 1082 units per mg in the presence of 1 mM fructose 1,6-bisphosphate. The enzyme was inhibited by NADPH, displaying an uncompetitive pattern. This pattern indicated that NADPH was a negative modifier of the enzyme. The role of L(+)-lactate dehydrogenase in controlling the end products of fermentation is discussed.

Bacteria, Anaerobic↗

PubMed1982

Stopped-flow kinetic studies on the D-lactate dehydrogenase from megasphera elsdenii.

The reaction of oxidized D-lactate dehydrogenase with D-lactate and reduced D-lactate dehydrogenase with pyruvate and oxygen was studied in a stopped-flow spectrophotometer. Oxidized enzyme was reduced in a triphasic manner by D-lactate at a variety of pH values. The fastest phase is thought to represent charge-transfer complex formation and is equal to an increase in long wavelength (510-700 nm) absorbance. The two slower phases seem to be due to flavin reduction. At pH 6 the proportion of the flavin reduction which proceeds by either phase is dependent upon the concentration of D-lactate. The reaction between reduced enzyme and oxygen is a simple second-order reaction. However, the oxidation of reduced D-lactate dehydrogenase by pyruvate is biphasic at pH 6 and monophasic at pH 8. At pH 8 there is a rapid increase in absorbance above 510 nm which is again ascribed to formation of a charge-transfer complex. These results are taken to indicate that D-lactate dehydrogenase may exist in two active conformations. At high pH the enzyme operates mainly through only one form while at low pH both forms participate in catalysis.

Hydrogen-Ion Concentration↗

PubMed1980

Oxygen effect in the radiolysis of proteins. I. Lactate dehydrogenase.

Dose survival curves of lactate dehydrogenase irradiated under aerobic and anaerobic conditions show a sensitizing effect of oxygen for the inactivation of the enzyme. The oxygen effect in the radiolysis of lactate dehydrogenase was analysed by gel filtration and polyacrylamide gel electrophoresis. Under anaerobic conditions the main reaction is the aggregation caused by covalent non-covalent interaction of the protein molecules. Oxygen prevents both kinds of aggregation and instead it enhances peptide chain breakage. Protein fragments became obvious only after reduction of the irradiated proteins and could be separated by electrophoresis.

Chromatography, Gel↗

PubMed1988

Ambiquitous behavior of rabbit liver lactate dehydrogenase.

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8-7.4) and ionic strength (20-50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO4(2-) and cations (Mg2+, Ca2+) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD+ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0-37 degrees C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.

Animals↗

PubMed1978

Improved column method for separating lactate dehydrogenase isoenzymes 1 and 2.

Lactate dehydrogenase (LD) isoenzymes 1 and 2 in human serum were separated on a column of diethylaminoethyl-Sephadex. Samples layered on mini-columns were eluted with buffered sodium chloride (100, 150, and 200 mmol/liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and their isoenzyme content was evaluated by electrophoresis on polyacrylamide gel. Results for column-fractionated LD-1 and LD-2 were expressed in two ways: LD-1/LD-2 ratios and total LD-1 + LD-2 activities. The former is a more specific indicator of myocardial infarction than the latter. Sera from 10 patients with acute myocardial infarction (increased creatine kinease isoenzyme MB activity) exhibited ratios in the range of 0.92 to 1.56, ratios for 10 patients without heart disease (normal creatine kinase MB) ranged from 0.33 to 0.69.

Chromatography, Ion Exchange↗

PubMed1988

Reverse micelles as a versatile medium for the study of lactate dehydrogenase in vitro.

Rabbit muscle lactate dehydrogenase has been solubilized in cationic reverse micelles of cetyltrimethyl ammonium bromide (CTAB) and isooctane-chloroform (1:1, V/V). The activity of the enzyme was notably affected by the change in water pool, pH, and concentration of the surfactant. Lactate dehydrogenase showed its full activity in this reverse micellar system in non-aqueous solvent under specific conditions at a Wo value of 30.55, pH 7.0, and 100 mM CTAB in comparison to the activity measured in aqueous system under optimum conditions. These results indicate that even the large and complex enzymes (M.W. hundred thousand and four subunits) can be solubilized in apolar solvents where they may retain their conformational integrity and oligomericity, i.e., optimum subunit-subunit interaction with maintenance of full activity.

Animals↗

PubMed2002

The role of lipid phase structure in the interaction of lactate dehydrogenase with phosphatidylserine. Activity studies.

Lactate dehydrogenase is one of the enzymes of the glycolytic path. It has been shown to be able to bind in vitro to cellular membranes. The presence of anionic phospholipids induces changes in the catalytic properties of the enzyme similar to those found when the enzyme is bound to natural membranes. In this study, a nonionic detergent (Tween 20), at concentrations not affecting the catalytic activity of LDH, was used to study the role of the lipid supra-molecular structure in the interaction between pig skeletal muscle lactate dehydrogenase and phosphatidylserine. Tween 20 changes the equilibrium of concentrations between the lipid supra-molecular forms. The detergent at the used concentration values did not alter the activity of the enzyme when it was used on its own, but did diminish the level of inhibition induced by the studied phospholipid. The obtained results showed that the interaction is reversible and that the bilayer structure of the lipid is essential for the inhibition.

Animals↗

PubMed1978

Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase.

1. The pH-dependencies of the binding of NADH and reduced nicotinamide--benzimidazole dinucleotide to pig heart cytoplasmic malate dehydrogenase and lactate dehydrogenase are reported. 2. Two ionizing groups were observed in the binding of both reduced coenzymes to lactate dehydrogenase. One group, with pKa in the range 6.3--6.7, is the active-site histidine residue and its deprotonation weakens binding of reduced coenzyme 3-fold. Binding of both coenzymes is decreased to zero when a second group, of pKa 8.9, deprotonates. This group is not cysteine-165.3. Only one ionization is required to characterize the binding of the two reduced coenzymes to malate dehydrogenase. The group involved appears to be the active-site histidine residue, since its ethoxycarbonylation inhibits the enzyme and abolishes binding of reduced coenzyme. Binding of either reduced coenzyme increases the pKa of the group from 6.4 to 7.4, and deprotonation of the group is accompanied by a 10-fold weakening of coenzyme binding. 4. Two reactive histidine residues were detected per malate dehydrogenase dimer. 5. A mechanism which emphasizes the homology between the two enzymes is presented.

Benzimidazoles↗

PubMed1979

Inhibition of lactate dehydrogenase activity from Dirofilaria immitis by suramin.

Lactate dehydrogenase from adult D. immitis was partially purified and characterized. The molecular weight of the enzyme was determined to be 130 000. The specific activity of the lactate dehydrogenase was 4.1 U/mg in the direction of NADH-oxidation and 0.38 U/mg protein in the direction of NAD-reduction. The Michaelis constants were determined to be 0.028 mM and 0.9 mM for NADH and NAD, respectively. Suramin was found to be a potent inhibitor of the lactate dehydrogenase activity. The inhibition constant was calculated to be 0.006 mM. The type of inhibition by suramin was competitive with respect to the cofactors. The Km-values for pyruvate and lactate were determined to be 0.3 mM and 11mM, respectively.

Animals↗

PubMed2002

Sol-gel encapsulation of lactate dehydrogenase for optical sensing of L-lactate.

Sol-gel encapsulation of lactate dehydrogenase and its cofactor can be employed as a disposable sensor for L-lactate. The sensor utilized the changes in absorbance or fluorescence from the reduced cofactor nicotinamide adenine dinucleotide (NADH) upon exposure to L-lactate. Although, problems such as diminished enzymatic activity and/or leaching of enzyme from the sol-gel matrix occurred, the sol-gel process is sufficiently mild to permit retention of enzymatic activity. The apparent activity of LDH in the sensor is at least 10% of that of the dissolved enzyme. The sensor has a linear dynamic range over the normal physiological L-lactate level and has a long-term storage stability of at least 3 weeks.

Biosensing Techniques↗

PubMed1992

An inquiry into the source of stereospecificity of lactate dehydrogenase using substrate analogues and molecular modeling.

Lactate dehydrogenase catalyzes the stereospecific hydride transfer to and from the re face of the nicotinamide coenzyme. The demonstrated probability of transfer to the si face of less than 2 x 10(-8) indicates that the free energy of any diastereotopic transition state leading to a si transfer must be over 10 kcal/mol greater than the free energy for transfer to or from the re face. The general notion of closed, desolvated active sites suggests the a priori hypothesis that steric hindrance prevents the nicotinamide ring from assuming a conformation that would lead to transfer of the pro-S hydrogen. In this paper we report that the probability of transfer of the pro-S proton is less than 9 x 10(-7) with 3-pyridinealdehyde adenine dinucleotide as coenzyme and less than 4 x 10(-7) during the lactate dehydrogenase catalyzed disproportionation of glyoxylate. Examination of the crystal structure of lactate dehydrogenase further suggests that steric exclusion does not enforce the extreme stereospecificity of the reaction. An electrostatic interaction with the macrodipole associated with the alpha 2F helix is suggested as a potential molecular source of the stereospecificity.

Animals↗

PubMed1979

The movement of pyruvate, lactate and lactate dehydrogenase into rabbit oviductal fluid.

Pyruvate, lactate and lactate dehydrogenase appeared linearly in 2 ml 0.9% NaCl recirculated through the rabbit oviduct for 4 h in vivo. In oviducts from rabbits injected 3 days previously with 100 i.u. hCG, the rate of appearance of all three constituents was considerably reduced. It is considered unlikely that the lactate dehydrogenase secreted brings about the interconversion of pyruvate and lactate in the oviduct lumen.

Animals↗

PubMed1975

Simultaneous separation of serum creatine kinase and lactate dehydrogenase isoenzymes by ion-exchange column chromatography.

Lactate dehydrogenase isoenzymes were partially separated by use of a previously described column technique for creatine kinase [Clin. Chem. 20, 36 (1974)]. Extracts of lactate dehydrogenase-rich tissues were used to evaluate column resolution. Samples layered on mini-columns containing DEAE-Sephadex were eluted with Tris-buffered sodium chloride (100 and 200 mmol/liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and their isoenzyme content was assessed by electrophoresis on polyacrylamide gel. Dehydrogenase isoenzymes 3, 4, and 5 were separated from isoenzymes 1 and 2, and the separation was tissue-specific and reproducible. The electrophoretic technique for isoenzymes 3, 4, and 5 gave values about 20% lower than did the column technique. Sera from 15 healthy laboratory technicians contained total lactate dehydrogenase, isoenzymes 1 and 2, and isoenzymes 3, 4, and 5 in the ranges 94 to 152, 34 to 64, and 38 to 75 U/liter, respectively. Activities of sera from 15 patients with acute myocardial infarction (total lactate dehydrogenase) ranged from 212 to 800 U/liter and lactate dehydrogenase isoenzymes 1 and 2 ranged from 138 to 628 U/liter. Lactate dehydrogenase and creatine kinase isoenzymes were rapidly and easily measured after being simultaneously separated. The procedure is specific and sensitive for following the post-infarct time course of changes in isoenzyme activities.

Chromatography, DEAE-Cellulose↗

PubMedDate not supplied

Cytophotometric analysis of reaction rates of succinate and lactate dehydrogenase activity in rat liver, heart muscle and tracheal epithelium.

Reaction rates of succinate and lactate dehydrogenase activity in cryostat sections of rat liver, tracheal epithelium and heart muscle were monitored by continuous measurement of formazan formation by cytophotometry at room temperature. Incubation media contained polyvinyl alcohol as tissue protectant and Tetranitro BT as final electron acceptor. Control media lacked either substrate or substrate and coenzyme. Controls were also performed by adding malonate (a competitive inhibitor of succinate dehydrogenase), pyruvate (a non-competitive inhibitor of lactate dehydrogenase), oxalate (a competitive inhibitor of lactate dehydrogenase) or N-ethylmaleimide (a blocker of SH groups). A specific malonate-sensitive linear test minus control response for succinate dehydrogenase activity was obtained in liver (1.6 mumol H2cm-3 min-1) and tracheal epithelium (0.8 mumol H2cm-3 min-1) but not in heart muscle. All variations in the incubation conditions tested did not result in a linear test minus control response in the latter tissue. Because the reaction was sensitive to malonate, it was concluded that the initial reaction rate was the specific rate of succinate dehydrogenase activity in heart muscle (9.1 mumol H2 cm-3 min-1). Test minus control reactions for lactate dehydrogenase activity were distinctly non-linear for all tissues tested. This appeared to be due to product inhibition by pyruvate generated during the reaction and therefore it was concluded that the appropriate control reaction was the test reaction in the presence of 20 mM pyruvate. The initial rate of the test minus this control was the true rate of lactate dehydrogenase activity. The lactate dehydrogenase activity thus found in liver parenchyma was 5.0 mumol of H2 generated per cm3 liver tissue per min.

Animals↗

PubMed1987

Peripartal changes in serum alkaline phosphatase activity and lactate dehydrogenase activity in dairy cows.

Peripartal serum alkaline phosphatase activity and lactate dehydrogenase activity were measured in 30 dairy cows in order to examine the association between retained fetal membranes and enzyme activity. Daily blood samples were obtained from pregnant cows, starting 15 days before the expected day of calving until eight days after parturition. Sera from 15 cows which retained fetal membranes longer than 24 hours and 15 cows which shed fetal membranes within six hours after parturition were analyzed for alkaline phosphatase and lactate dehydrogenase enzyme activities. Mean alkaline phosphatase enzyme activities ranged from 15.93 to 32.6 U/L in retained and nonretained placenta cows. There was a trend towards higher serum alkaline phosphatase activities in retained placenta cows but the differences were not significant among the groups (P greater than 0.05). Mean lactate dehydrogenase activities ranged from 307.2 to 438.86 U/L in nonretained and retained placenta cows. Lactate dehydrogenase enzyme activities in nonretained and retained placenta cows were similar (P greater than 0.05). The alkaline phosphatase and lactate dehydrogenase enzyme activities peaked at the time of parturition in both groups. However, the differences in alkaline phosphatase and lactate dehydrogenase activities on different days within non-retained and retained placenta cows were significant (P less than 0.05). Results indicate that prepartal changes in alkaline phosphatase and lactate dehydrogenase enzyme activities are not predictive of placental retention postpartum.

Alkaline Phosphatase↗

PubMed2003

Activities of enzymes in the malate-aspartate shuttle and the isoenzyme pattern of lactate dehydrogenase in plasma and peripheral leukocytes of lactating Holstein cows and riding horses.

The activities of the enzymes involved in the malate-aspartate shuttle and lactate dehydrogenase (LDH) and the pattern of the isoenzymes of LDH were determined in plasma and peripheral leukocytes of lactating Holstein cows and thoroughbred riding horses as representative herbivorous animals. In the horse plasma, LDH activities were significantly lower and AST activities were significantly higher than those in the cow plasma. The specific activities of cytosolic malate dehydrogenase (MDH), LDH and AST in the horse leukocytes were higher than those in the cows. The cytosolic ratio of MDH/LDH activity (ML ratio) in the horse leukocytes was significantly lower than that in the cow leukocytes owing to significantly higher activities of LDH. The ML ratio was considered to reflect the difference in energy metabolism in leukocytes between cows and horses. The plasma LDH isoenzyme patterns of cow and horse showed the characteristic as herbivorous animals with dominance of LDH-1, -2 and -3. The LDH isoenzyme patterns with dominance of LDH-3 and -4 in the horse leukocytes were remarkably different from those in the cow leukocytes. There were significant differences in activities of malate-aspartate shuttle enzymes, ML ratio and LDH isoenzyme patterns in the cytosolic fractions of leukocytes between the lactating cows and the riding horses.

Amino Acid Oxidoreductases↗