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PubMed1982

Lactate dehydrogenase isoenzyme 1 in the diagnosis of myocardial infarction.

A new immunochemical method for the measurement of lactate dehydrogenase isoenzyme 1 (LDH-1) was used on 113 consecutively admitted patients with suspected myocardial infarction. Using WHO's criteria and our routine program 49 were classified as having myocardial infarction and 64 as having no myocardial infarction. LDH-1 was better than total lactate dehydrogenase is discriminating between patients with and without myocardial infarction (p less than 0.02), and the efficiency (the number of correct classifications divided by the total number of analyses) on day 2-3 equalled that from the literature for CK-MB on day 1. Total lactate dehydrogenase and LDH-1 are supplemental to creatine kinase (CK) and CK-MB, while aspartate aminotransferase may be omitted in the diagnosis of myocardial infarction. Determination of the ratio of LDH-l to total lactate dehydrogenase offered to no advantage.

Aged↗
PubMed1976

Evidence for the lack of feedback regulation of gene activity and for the absence of subunit exchange between lactate dehydrogenase tetramers in vivo.

Frog and rat lactate dehydrogenase (LDH, L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) isozymes, highly purfied by affinity chromatography, were injected into newly fertilized from (Rana pipiens) eggs to study the effect of the injected isozymes on the expression of the LDH genes of the developing frog embryo. From these experiments three conclusions can be drawn: (i) homologous and heterologous LDH, even when present in more than twice the normal amount of LDH, does not play any role in the switching on or off of the frog LDH genes or in regulating the level of gene activity. No evidence was found in support of any feedback regulation of LDH synthesis by the LDH molecule itself. (ii) Injected rat LDH is very stable in the frog embryo, and the contrary to what can be demonstrated in vitro, all the isozymes show the same stability. No evidence was found for selective degradation of any of the injected five isozymes. (iii) The fact that the injected isozymes disappear 12 days after the injection without forming hybrid molecules between themselves or with the stored or newly synthesized frog LDH is evidence in favor of the hypothesis that the tetramer is the degradative unit of LDH, not the monomer as others have postulated. The LDH tetramers do not dissociate the recombine under the physiological conditions present in frog cells.

Animals↗
PubMed1968

D-lactate specific pyridine nucleotide lactate dehydrogenase in animals.

A survey of representative invertebrates has revealed the presence of pyridine nucleotide-linked (D)-lactate dehydrogenase in a number of groups. All species studied contained either D(D)-or (L)L-lactate dehydrogenase, but no species contained both enzymes. The (D)-lactate dehydrogenase from Limulus polyphemus has been purified and has a molecular weight of 65,000.

Animals↗
PubMed1994

The isoenzyme forms of lactate dehydrogenase from the testes of Uromastix hardwickii.

Lactate dehydrogenase was purified from testes of Uromastix hardwickii. The enzyme did not bind to DEAE-Sepharose at pH 7.2. A gel electrophoretic study of the crude enzyme showed the presence of three isoenzymes. The pH optima for pyruvate reduction and lactate oxidation were 7.0 and 9.5, respectively. The purified enzyme showed a single band after SDS-PAGE corresponding to a molecular weight of 34 KDa. The Km values for pyruvate, NADH, lactate and NAD+ were 0.019, 0.045, 9.0 and 0.011 mM, respectively. Pre-heating of the enzyme showed that it was stable up to 70 degrees C.

Animals↗
PubMed1989

Lactate dehydrogenase from adult Setaria digitata (Nematoda: Onchocercidae).

Lactate dehydrogenase of Setaria digitata exhibited an optimum pH of 7.0-8.0 and showed resistance to high temperature. The inhibition/activation of various anions differed in both the forward and backward directions. Filarin (a drug used in Siddha medicine) and diethylcarbamazine (DEC) inhibited pyruvate reduction rather than lactate oxidation. High pyruvate reduction:lactate oxidation at Vmax and Vmax/Km favoured pyruvate reduction in vivo. The enzyme exists as isozymes (four in the female and three in the male) and their separation depended on the percentage of gel and on pH. The mobility of the 700 X g supernatant fraction in the gel was less than that of the 10,000 X g supernatant.

Animals↗
PubMed1991

Limited proteolysis of lactate dehydrogenase from porcine heart with trypsin: characterization and reactivation of the fragments.

The ternary complex formed by native lactate dehydrogenase (LDH) from porcine heart, NAD+ and sulfite, was digested with trypsin over a period of 12-16 h3. After removal of the ligands and residual native lactate dehydrogenase by ion exchange chromatography dimers were obtained which were almost inactive. The dimers were lacking a hexapeptide at the N-terminus; however, the secondary structure was the same as that of native lactate dehydrogenase. The circular dichroism spectra showed a dependence on temperature which suggested an equilibrium of two different structural states. The reaction of antibodies against native porcine heart LDH with the dimers restored the catalytic activity, and subsequently the dimers behaved similarly to the native enzyme. Addition of 1 M phosphate or NAD-sulfite to the dimers restored 80-90% of the catalytic activity. It could be demonstrated that the behavior of the reactivated dimers, in contrast to that of the inactive dimers, was similar to the behavior of native lactate dehydrogenase. For instance, ultracentrifugal analysis showed that dimers reactivated with NAD-SO3- were associated to give tetramers. The reaction of antibodies against native LDH with the dimers reactivated with NAD-SO3- demonstrated that the native LDH and the dimers have the same surface determinants.

Amino Acid Sequence↗
PubMed1976

Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei.

The fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue-Sephadex-G-200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000-135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 insodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 degrees C to 70 degrees C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6-bisphosphate. In the presence of 20 muM fructose 1,6-bisphosphate a Km of 1.0 mM for pyruvate was obtained, whereas fructose 1,6-bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6-phosphogluconate are strong inhibitors. Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. casei ATCC 393 L (+)-lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross-react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 - L. casei var. alactosus NCDO 680 greater than L. casei UQM 95 greater than L. plantarum ATCC 14917.

Cations, Divalent↗
PubMed1988

Serum lactate dehydrogenase isoenzyme 1 as a marker of testicular germ cell tumor.

Serum lactate dehydrogenase isoenzyme 1 activity was determined repeatedly in 21 men with testicular germ cell tumors in connection with orchiectomy and in 25 without neoplasia who underwent exploration of the testis. The highest level in the men without malignancy was 109 units per 1. and a higher pre-orchiectomy level was found in 15 of the tumor patients: 7 of 11 with seminoma and 8 of 10 with nonseminomatous tumors. In the patients with stage 3 disease serum lactate dehydrogenase isoenzyme 1 was increased more often and the activity was higher than in the stage 1 and 2 cancer patients. Within 1 month after orchiectomy the initially increased level decreased to less than 109 units per 1. in 8 of 10 patients with a stage 1 tumor and it remained higher in 5 with stage 2 or 3 disease. Serum lactate dehydrogenase isoenzyme 1 seems to be a useful marker of testicular germ cell tumor.

Adult↗
PubMed1977

Substrate-induced conformational changes in lactate dehydrogenase. Proteolysis of the immobilized enzyme in the presence of specific substrates.

We report here a new approach to the study of the conformation of enzymes in the presence of specific substrates. Rabbit muscle lactate dehydrogenase was attached to CL-Sepharose via a cleavable spacer arm (-NH-(CH2)6NHCO(CH2)2SS(CH2)2CO-). The bound lactate dehydrogenase was digested with subtilisin BPN' in the presence of substrates of lactate dehydrogenase. The use of a flow system permits the maintenance of saturating levels of substrates. Proteolysis was followed by loss of activity of the enzyme column. The time course of proteolysis in the presence of either NADH, NAD+, or pyruvate alone did not differ from the control. However, when NADH and pyruvate were present simultaneously, the enzyme became more susceptible to proteolysis. The initial rate of proteolysis was increased by 40%. The abortive ternary complex (lactate dehydrogenase - NAD+ - pyruvate) also showed an increase in susceptibility to proteolysis. These findings clearly show that the productive ternary complex (lactate dehydrogenase - NADH - pyruvate) is conformationally different from the apoenzyme and binary complexes under optimal catalytic conditions.

Enzymes, Immobilized↗
PubMed1995

Cloning, nucleotide sequence, and transcriptional analysis of the Pediococcus acidilactici L-(+)-lactate dehydrogenase gene.

Recombinant plasmids containing the Pediococcus acidilactici L-(+)-lactate dehydrogenase gene (ldhL) were isolated by complementing for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase-pyruvate formate lyase double mutant. The nucleotide sequence of the ldhL gene predicted a protein of 323 amino acids showing significant similarity with other bacterial L-(+)-lactate dehydrogenases and especially with that of Lactobacillus plantarum. The ldhL transcription start points in P. acidilactici were defined by primer extension, and the promoter sequence was identified as TCAAT-(17 bp)-TATAAT. This sequence is closely related to the consensus sequence of vegetative promoters from gram-positive bacteria as well as from E. coli. Northern analysis of P. acidilactici RNA showed a 1.1-kb ldhL transcript whose abundance is growth rate regulated. These data, together with the presence of a putative rho-independent transcriptional terminator, suggest that ldhL is expressed as a monocistronic transcript in P. acidilactici.

Amino Acid Sequence↗
PubMedDate not supplied

[Antipeptide antibodies specific for the N-terminal site of lactate dehydrogenase from swine muscle].

Antipeptide antisera were obtained after immunization of rabbits with N-terminal peptide of pig muscle lactate dehydrogenase-5 containing the enzyme antigen determinant. The N-terminal peptide (1-32 amino acids) was conjugated with bovine blood serum albumin. Specific antipeptide antibodies were isolated and purified using immunosorbent involving the antigen--lactate dehydrogenase-5. Immunodiffusion procedure and immunoenzymatic assay showed that the antipeptide antibodies obtained were specific towards conjugate, N-terminal peptide and the native pig muscle lactate dehydrogenase-5. The antipeptide antibodies towards N-terminal fragment of lactate dehydrogenase did not affect the enzymatic activity.

Animals↗
PubMed1998

Quantification of relative expression of genes with homologous sequences using fluorescence-based single-strand conformation polymorphism analysis--application to lactate dehydrogenase and cyclooxygenase isozymes.

The combination of reverse transcribed-PCR and fluorescence-based single strand conformation polymorphism analysis has been proposed for the quantitative determination of ratio of mRNA molecules with homologous sequences. We applied this procedure to lactate dehydrogenase subunits M and H, and cyclooxygenase 1 and 2. We designed fluorescence labeled common PCR primers in the sequences highly homologous between two subunit and isozyme cDNAs, and performed reverse transcribed-PCR and fluorescence-based single strand conformation polymorphism analysis. PCR efficiency was almost the same for the different target sequences, so analysis of mixtures of known amounts of lactate dehydrogenase M and H revealed linear and precise proportions of lactate dehydrogenase M mRNA. It was shown that template concentrations and number of PCR cycles did not affect the determination of proportions of lactate dehydrogenase M to total lactate dehydrogenase. The procedure was applicable to a determination of cyclooxygenase-2 proportion; furthermore, the present procedure could be easily applied to investigation of expression levels of genes encoding mRNAs with homologous sequences.

Amino Acid Sequence↗
PubMed1992

Cloning and overexpression of the Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene in Escherichia coli:purification and characterization of the recombinant enzyme.

The Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene was amplified by the polymerase chain reaction and cloned into an Escherichia coli expression plasmid pKK223.3. Attempts to clone the full-length chromosomal DNA encoding D-lactate dehydrogenase from a partial Sau3AI lambda phage library or an enriched clone bank in E. coli were unsuccessful. The recombinant plasmid pKBULDH containing the amplified gene overexpressed D-lactate dehydrogenase (greater than 30% of total soluble protein) following induction of the tac promotor with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by two chromatographic steps with 76% recovery of enzyme activity. All the properties of the recombinant protein, e.g., optimum pH and temperature, Km and k(cat) for pyruvate as well as for other 2-oxo acids and the subunit structure were identical to the wild-type enzyme.

Amino Acid Sequence↗
PubMedDate not supplied

[Different character of zinc ion binding with lactate dehydrogenase M4 in diluted and concentrated solutions].

The study of the melting characteristics of lactate dehydrogenase M4 and Zn/lactate dehydrogenase at pH 6.5-8.5 by microcalorimetric methods shows that in dilute solutions of the enzyme (0.05% lactate dehydrogenase solution) one Zn ion causes destabilisation of enzyme structure, attended by a decrease of melting temperature throughout the entire pH interval under study. Concentrated solutions of the enzyme (0.4% lactate dehydrogenase solution) are a seat of such conformational changes of the latter which affect the character of Zn ions binding depending on pH.

Animals↗
PubMed1990

Use of lactate dehydrogenase release to assess changes in culture viability.

This study reports the use of lactate dehydrogenase release to monitor changes in culture viability in flask culture and fixed bed, porosphere bioreactor systems. Lactate dehydrogenase release shows good agreement with increase in non-viable cell numbers and decline in glucose utilisation in flask cultures. Studies with the immobilised system show that lactate dehydrogenase release can detect loss of viability which is not always indicated by a decrease in glucose utilisation. The data show that culture viability in a repeated-feed-and-harvest system is influenced markedly by both a) the medium change regime itself and b) the use of an immobilised bioreactor compared to a flask system for the same medium change regime.

Animals↗
PubMed1985

Monoclonal antibodies against the membrane-bound, flavin-linked D-lactate dehydrogenase of Escherichia coli: preparation, characterization, and use in immunoaffinity chromatography.

Three mouse hybridoma cell lines are described that produce monoclonal antibodies directed against the membrane-bound, flavin adenine dinucleotide linked D-lactate dehydrogenase of Escherichia coli. In contrast to polyclonal antibodies produced in rabbits, none of the monoclonal antibodies inhibits enzyme activity. Immunoblots of D-lactate dehydrogenase proteolytic fragments indicate that each antibody is directed against a different region of the molecule. One monoclonal antibody, 1B2a, reacts with native, undigested D-lactate dehydrogenase only and is used to purify the enzyme in a single step. The protocol involves chromatography of a Triton X-100 extract of membrane vesicles containing D-lactate dehydrogenase on a column made with the monoclonal antibody coupled to a solid support. After the column is washed free of unadsorbed protein, elution at high pH in the presence of guanidine hydrochloride yields a fraction containing highly purified, catalytically active D-lactate dehydrogenase.

Animals↗