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PubMed1981

Purification of lactate dehydrogenase isoenzyme-5 from human liver.

We present a method for preparing human liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase; EC 1.1.1.27) isoenzyme-5 by sequential ion-exchange chromatography, general-ligand (AMP analog) affinity chromatography, and preparative isoelectric focusing. The yield ws 40%, with a 493-fold purification. The final specific activity was 458 kU per gram of protein. The preparation contained less than 0.2% of lactate dehydrogenase isoenzyme-4, was homogeneous by agarose gel electrophoresis and also by polyacrylamide gel electrophoresis at pH 8.9 and 6.9, and showed one major protein band (containing all the enzyme activity) and one minor anodic contaminant (containing no enzyme activity) by analytical isoelectric focusing. The enzyme had a mean pI value of 9.59 (SD 0.04) (n = 5) at 5 degrees C. By comparison, the pI value of a preparation of rabbit lactate dehydrogenase-5 was 9.16 (5 degrees C).

Chromatography, Affinity↗
PubMed1998

Modulation of DNA polymerases alpha, delta and epsilon by lactate dehydrogenase and 3-phosphoglycerate kinase.

Literature documents that glycolytic enzymes (among them lactate dehydrogenase and 3-phosphoglycerate kinase) can reside in nuclei of mammalian cells and exert functions in DNA replication, transcription and DNA repair, in addition to their role as catalysts in the cytoplasm. Transfer of glycolytic enzymes to cell nuclei requires modification, for example phosphorylation. We studied the effects of phosphorylated lactate dehydrogenase and 3-phosphoglycerate kinase on (i) UV-induced DNA repair, using permeabilized human fibroblasts, and (ii) in vitro DNA synthesis catalyzed by purified DNA polymerases alpha, delta, and epsilon from proliferating rat liver. (i) Phosphorylated lactate dehydrogenase stimulated UV-induced DNA repair synthesis in normal fibroblasts in a dose-dependent manner; the unphosphorylated enzyme slightly inhibited. In repair-deficient xeroderma pigmentosum fibroblasts reparative synthesis was not enhanced whether lactate dehydrogenase was phosphorylated or not, indicating that reparative DNA synthesis must be possible in order to be stimulated. (ii) Activity of purified DNA polymerases alpha, delta, and epsilon was differentially stimulated or inhibited, according to the phosphorylation status of lactate dehydrogenase. DNA polymerases were also modulated by 3-phosphoglycerate kinase, depending on the primer-templates used which were gapped DNA (mimicking a repair mode of DNA synthesis) or single-stranded M13 DNA (representing the replicative mode of DNA synthesis). Since glycolytic enzymes in cell nuclei retain binding ability for their cofactors, cytoplasmic substrates and inhibitors, a regulatory linkage might exist between the energy state of a cell and its replicative and reparative functions.

Animals↗
PubMed1993

A 19F-NMR study of the membrane-binding region of D-lactate dehydrogenase of Escherichia coli.

D-Lactate dehydrogenase (D-LDH) is a membrane-associated respiratory enzyme of Escherichia coli. The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65,000, and requires lipids or detergents for full activity. We used NMR spectroscopy to investigate the structure of D-LDH and its interaction with phospholipids. We incorporated 5-fluorotryptophan (5F-Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide-directed mutagenesis, and studied the 5F-Trp-labeled enzymes using 19F-NMR spectroscopy. In this way, information was obtained about the local environment at each native and substituted tryptophan site. Using a nitroxide spin-labeled fatty acid, which broadens the resonance from any residue within 15 A, we have established that the membrane-binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region. This conclusion is strengthened by the results of 19F-NMR spectroscopy of wild-type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin-labeled fatty acid. These experiments indicate that 9-10 Phe and 3-4 Tyr residues are located near the lipid phase.

DNA Mutational Analysis↗
PubMed2001

Selective active site inhibitors of human lactate dehydrogenases A4, B4, and C4.

Human lactate dehydrogenases (LDH-A4, -B4, and -C4) are highly homologous with 84-89% sequence similarities and 69-75% amino acid identities. Active site residues are especially conserved. Gossypol, a natural product from cotton seed, is a non-selective competitive inhibitor of NADH binding to LDH, with K(i) values of 1.9, 1.4, and 4.2 microM for LDH-A4, -B4, and -C4, respectively. However, derivatives of gossypol and structural analogs of gossypol in the substituted 2,3-dihydroxy-1-naphthoic acid family exhibited markedly greater selectivity and, in many cases, greater potency. For gossypol derivatives, greater than 35-fold selectivity was observed. For dihydroxynaphthoic acids with substituents at the 4- and 7-positions, greater than 200-fold selectivity was observed. Inhibition was consistently competitive with the binding of NADH, with dissociation constants as low as 30 nM. By comparison, a series of N-substituted oxamic acids, which are competitive inhibitors of the binding of pyruvate to LDH, exhibited very modest selectivity. These results suggest that substituted dihydroxynaphthoic acids are good lead compounds for the development of selective LDH inhibitors. Selective inhibitors of LDH-C4 targeted to the dinucleotide fold may hold promise as male antifertility drugs. Selective inhibitors of LDH-A4 and -B4 may be useful for studies of lactic acidemia associated with ischemic events. More broadly, the results raise the question of the general utility of drug design targeted at the dinucleotide binding sites of dehydrogenases/reductases.

Amino Acid Sequence↗
PubMed1981

Influence of hemolysis on the serum lactate dehydrogenase-1/lactate dehydrogenase-2 ratio as determined by an accurate thin-layer agarose electrophoresis procedure.

The accuracy of our lactate dehydrogenase (EC 1.1.1.27) isoenzyme procedure, based on electrophoresis in thin-layer agarose, has previously been assessed (Clin. Chem. 22: 1995, 1976). We now show that the method is similarly accurate and precise for the specific measurements of lactate dehydrogenase isoenzyme-1 and -2. We have also determined the lactate dehydrogenase isoenzyme pattern in peripheral erythrocytes and have found that lactate dehydrogenase-1 is usually the isoenzyme present in greatest concentration. Therefore, hemolysis may increase lactate dehydrogenase-1 in serum, affecting the lactate dehydrogenase isoenzyme ratio used in the diagnosis of myocardial infarction. This conjecture has been confirmed by in vitro experiments, and in a small group of patients with intra-vascular hemolysis.

Clinical Enzyme Tests↗
PubMedDate not supplied

[Antigenic properties of lactate dehydrogenase isoenzymes of various biological types using antipeptide antibodies to a fragment of the active center of swine lactate dehydrogenase-M4].

Antigenic properties of human, pig, rabbit and rat lactate dehydrogenase (LDH) M4-isozyme as well as of human LDH-C4 were studied using antipeptide antibodies against pig LDH-M4 fragment (180-214). Amino acid sequence 180-214, containing His-195, which is involved in the active site of LDH isozymes, proved to be total antigenic determinant only for human and pig M4-isozymes. Amino acid sequence of total antigenic determinant did not allow any substitutions of essential amino acid residues. His-195, participating in substrate binding, was not involved immediately in reactions with antibodies as shown by means of chemical modification of pig M4-isozyme with diethyl pyrocarbonate and after production of antibodies against the modified isoenzyme.

Animals↗
PubMed1991

Lactate Dehydrogenase in Oryza sativa L. Seedlings and Roots: Identification and Partial Characterization.

A lactate dehydrogenase activity is present in rice (Oryza sativa L.) seedlings and roots. Under aerobic conditions, lactate dehydrogenase activity is barely detectable in rice seedlings and is very low in rice roots. In 30 day old roots, the activity is increased two to three times by an anoxic or hypoxic treatment and can be detected on immunoblots by an antiserum raised against barley lactate dehydrogenase. The activity present in aerobic seedlings was partially purified. The native enzyme has a molecular mass of 160 kilodaltons, and is a tetramer of 2 subunit (38 and 39 kilodaltons) randomly associated. Studies of substrate specificity, native gel electrophoresis, and immunoblot analysis indicate that the partially purified enzyme is a typical lactate dehydrogenase. However, no increase of lactate dehydrogenase activity or protein was observed in seedlings transferred to anoxia.

Journal Article↗
PubMed1977

Structural adaptations of lactate dehydrogenase isozymes.

The three-dimensional structures of dogfish M4 (muscle) and pig H4 (heart) lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) have been determined and correlated with the amino acid sequences of the dogfish M4, pig M4, pig H4, chicken M4, and chicken H4 lactate dehydrogenase isozymes. These results have been related to the known differences of physicochemical properties between the M and H lactate dehydrogenase isozymes. By far the largest structural alterations occur in the transition between the "apo" and "ternary complex" conformational states of the enzyme rather than between species or isozymes. The major catalytic difference can be explained by a replacement of alanine (in the M chain) with a glutamine (in the H chain) in the vicinity of the binding site of the coenzyme phosphates. The known immunological differentiation of the M and H isozymes is consistent with the differences in their amino acid sequences.

Adenosine↗
PubMed1981

Stability of lactate dehydrogenase. II. Hybrids and geometric isomers.

Hybrids of lactate dehydrogenases from pig heart and muscle and from chicken heart and pig heart were obtained by the freeze-thaw method [1,2]. Ion-exchange chromatography of the resulting mixtures of hybrids yielded unusual elution patterns, i.e., the 2 + 2 hybrids (HP2MP2 and HC2HP2) were eluted in two separate peaks. These subforms were concluded to result from partial resolution of the three geometric isomers. The hybrids of chicken heart and pig heart lactate dehydrogenase showed three distinct levels of stability. The characteristics temperatures of denaturation were 61.5 degrees C for HP4, HCHP3 and HC2HP2I; 71 degrees C for HC2HP2II and HC3HP and 76.5 degrees for HC4. The resistance towards thermal denaturation thus seemed to be governed by the least stable dimer within the tetrameric enzyme. The arrangement of stabilities of the dimers was in excellent agreement with the number of additional ion pairs between Arg241 (chicken) and Asp57 (chicken and pig) [3] within the Q-contact areas. The rate-determining step of thermal denaturation of lactate dehydrogenase was concluded to comprise the distortion or dissociation of one or two Q-contacts of the tetramer.

Animals↗
PubMed1977

Partial purification and characterization of L-lactate dehydrogenase isozymes from sweet potato roots.

Lactate dehydrogenase [L-lactate: NAD oxidoreductase, EC 1.1.1.27] was isolated from sweet potato root tissues. Two species of the enzyme (isozymes I and II) were separated by DE-52 cellulose column chromatography from healthy, cut, and black-rot diseased tissues. Isozymes I and II were purified from healthy and diseased tissues, respectively. Reduction of pyruvate by NADH with either isozyme I or II was inhibited by pyruvate at high concentrations, by NAD+ and by several mononucleotides. Isozyme I was inhibited by a lower concentration of adenine nucleotide than isozyme II, and Km for pyruvate was increased markedly at acidic pH in the case of isozyme I, but only slightly in the case of isozyme II. The molecular weights of both isozymes were determined to be 150,000 and they were found to be charge isomers by polyacrylamide gel electrophoresis. The enzyme activity increased in response to infection by black-rot fungus but decreased in response to cutting.

Hydrogen-Ion Concentration↗
PubMed1990

Diagnostic efficiency of lactate dehydrogenase isoenzymes in serum after acute myocardial infarction.

Diagnostic efficiency of lactate dehydrogenase isoenzymes were studied in 117 consecutive patients admitted with some symptoms of acute myocardial infarction. The results of lactate dehydrogenase isoenzyme tests were not available to the physicians, who diagnosed the patients according to criteria based on clinical symptoms, electrocardiographic findings and changes in three serum enzymes. Acute myocardial infarction was diagnosed in 41 patients. The diagnostic efficiency of lactate dehydrogenase isoenzyme 1 and the various ratios between this and the other isoenzymes were compared using receiver operating characteristic curves and logistic discriminant analysis. Lactate dehydrogenase isoenzyme 1 was the best parameter on the second day after admission. On that day, calculating the various ratios between isoenzyme 1 and the other isoenzymes did not improve discrimination.

Adult↗
PubMed1996

Glutamate-induced efflux of protein, neuron-specific enolase and lactate dehydrogenase from a mesencephalic cell culture.

A mixed mesencephalic cell culture damaged by glutamate was used as a model to study the efflux of lactate dehydrogenase and neuron-specific enolase from neuronal cells into the culture medium. Glutamate toxicity was induced in sister cultures by 15 min exposure to 100 mumol/l glutamate in a Ca2+ containing salt solution. Cell injury was monitored 24 h later by measuring the lactate dehydrogenase activity and the neuron-specific enolase content in the cells and in the culture medium. The neuronal cell damage is reflected by an efflux of neuron-specific enolase and lactate dehydrogenase from the cells and an increase of lactate dehydrogenase catalytic activity concentration and neuron-specific enolase mass concentration in the culture medium. It was found that the efflux fraction calculated from estimations of the cells was clearly higher than the efflux fraction calculated from estimations of the amount of enzymes found in the culture medium. Calculations of the recovery of lactate dehydrogenase and neuron-specific enolase and experiments designed to study the efflux of lactate dehydrogenase and neuron-specific enolase during incubation and washing showed that higher amounts of neuron-specific enolase are released than lactate dehydrogenase. A close correlation was found between the glutamate-induced changes of the neuron-specific enolase efflux fraction, based on enzyme determinations of the cells, and the change of the microscopically counted neuron-specific enolase immunoreactive cell numbers. This indicates that the determination of the neuron-specific enolase efflux fraction (cells) is an accurate and sensitive marker of damaged neurons. The lactate dehydrogenase efflux fraction seems to be less sensitive for the quantitation of neuronal cell damage; in addition, it depends not only on the neuronal damage but also on the proportion of neurons in the cell culture.

Animals↗
PubMed1987

The engineering of a more thermally stable lactate dehydrogenase by reduction of the area of a water-accessible hydrophobic surface.

A site-directed mutant of Bacillus stearothermophilus lactate dehydrogenase (lactate:NAD+ oxidoreductase, EC 1.1.1.27) has been engineered in which the conserved hydrophobic residue isoleucine-250 has been replaced by the more hydrophilic residue asparagine. This isoleucine forms a large part of a water-accessible, hydrophobic surface in the active site of the apo-enzyme which is covered by the B-face of the nicotinamide ring when coenzymes are bound. Reduction in the area of this hydrophobic surface results in the mutant tetramer being more thermally stable than the wild-type enzyme.

Binding Sites↗
PubMed1969

The foetal development of lactate dehydrogenase isoenzymes, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from human striated muscle.

1. Human foetal skeletal muscles involved in support and in periodic contractility were studied for their content of total extractable lactate dehydrogenase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities as well as for the relative distribution of lactate dehydrogenase isoenzymes. 2. During foetal development a linear steady increase in total lactate dehydrogenase activity as well as a linear decrease in the H/M sub-unit ratio of the isoenzymes was found. 3. No significant changes were found in the activities of the enzymes of the hexose monophosphate shunt (C-6 oxidation). 4. The changes found suggest a steady increased synthesis of lactate dehydrogenase M-sub-units in human skeletal muscles during foetal development. 5. The weekly changes in the total lactate dehydrogenase activity and in lactate dehydrogenase isoenzymes are lower in muscles involved in support than in those involved in periodic contractility. 6. These findings, together with the literature available, are consistent with the morphological fact that foetal development of skeletal muscles mostly concerns the white muscle fibres and not the red muscle fibres.

Female↗
PubMed1990

Kinetic formulations for the reduction of ketomalonate by lactate dehydrogenase.

Initial rate kinetic studies of lactate dehydrogenase with ketomalonate and NADH as substrates suggest that this enzymatic system is adapted to a rapid equilibrium ordered bi-bi ternary complex mechanism. The application of the reaction product inhibition method reveals the existence of the enzyme-NADH-hydroxymalonate and enzyme-NAD(+)-ketomalonate abortive complexes. This kinetic behaviour is confirmed by the differential inhibition induced by several alternate products on the pyruvate-lactate dehydrogenase-NADH and ketomalonate-lactate dehydrogenase-NADH systems.

Animals↗
PubMed1975

Localization of D-lactate dehydrogenase in membrane vesicles prepared by using a french press or ethylenediaminetetraacetate-lysozyme from Escherichia coli.

The localization of D-lactate dehydrogenase in membrane vesicles prepared from Escherichia coli was studied using antibody against the purified enzyme. The activity of D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by using a French press were completely inhibited by this antibody, suggesting that the enzyme is localized on the outside of these vesicles. This and previous results (Futai, 1974) strongly indicate the inversion of these vesicles. The D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by treatment with ethylenediaminetetraacetate-lysozyme were inhibited about 15% by the antibody, whereas proline transport of the vesicles was insensitive to antibody. These results suggest that most of the membrane vesicles have D-lactate dehydrogenase on the inside of the membrane and that such vesicles transport amino acids. This essentially confirms the results of Short, Kaback, and Kohn (1975). However, unlike them we observed that a small but significant portion of activity was sensitive to the antibody as shown above. This portion may represent the completely inverted vesicles in the preparation. Ferricyanide reductase activity cannot be detected in spheroplasts, but about 30 to 50% of the total was detected in membrane vesicles prepared by treatment with ethylenediaminetetraacetate. This confirms our previous findings with membrane prepared by a slightly different procedure. It is concluded that in these vesicles about half the reactive sites for ferricyanide are moved from inside to outside the membrane, whereas 85% of the D-lactate dehydrogenase remains inside the membrane.

Antibodies, Bacterial↗
PubMed1996

Synovial fluid levels of lactate dehydrogenase in patients with total knee arthroplasty.

Synovial fluid lactate dehydrogenase determination may offer a valuable preoperative assessment of polyethylene wear in aseptic total joint arthroplasty. Particulate ultrahigh-molecular-weight polyethylene may result in cell lysis and release of intracellular enzymes. This study tests the possible relationship between lactate dehydrogenase levels and particle wear in synovial fluid. The lactate dehydrogenase level may be used as a marker of joint inflammation from polyethylene wear.

Biocompatible Materials↗