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PubMed1992

Oxalate production from glyoxylate by lactate dehydrogenase in vitro: inhibition by reduced glutathione, cysteine, cysteamine.

Lactate dehydrogenase is known to act as a dismutase converting glyoxylate to oxalate and glycolate. LDH (sources: human erythrocytes, human plasma; rabbit muscle; rat liver) activity was assayed at 340 nm using glyoxylate (5.0 mmol/l) and NADH. The LDH activity (approx. % of control) in all the cases decreased respectively in the presence of 5.0 and 10.0 mmol/l of cysteine (45 and 20), cysteamine (45 and 20), and GSH (55 and 30). This decrease in LDH activity resulted in decreased oxalate production from glyoxylate (0.5 mmol/l). A 50% inhibition in oxalate production was observed in presence of 0.3 mmol/l cysteine, 0.35 mmol/l cysteamine, and 2.0 mmol/l GSH. The results suggest that the net LDH activity towards oxalate production may be regulated by the free SH-groups in the cell. This possibility needs evaluation as a tool to lower endogenous oxalate production and the associated risk of stone formation.

Animals↗

PubMed1986

An immunoglobulin G inhibiting lactate dehydrogenase activity.

We found extremely low (6 U/l) serum lactate dehydrogenase (LDH, EC 1.1.1.27) activity in a patient with uterine myoma. The patient's serum inhibited purified LDH isoenzymes. One milliliter serum neutralized 73 U purified LDH-1 isoenzyme, equivalent to 300-500-fold the amount of LDH in 1 ml of normal serum. The serum inhibitor was purified by ordinary procedures using chromatographies and identified as IgG which contains both kappa- and lambda-chains. The IgG is very unique in showing higher affinity to the H- than M-subunit of the LDH tetramer, in contrast with the IgGs already reported. After removal of the myoma, the anti-LDH activity gradually decreased with a half-life of 20 days corresponding to that of IgG and finally almost disappeared. This indicates a possibility that the myoma cells produce some factors such as B-cell growth and differentiation factors.

Chromatography↗

PubMed1978

Mechanistic study of the addition of pyruvate to NAD+ catalyzed by lactate dehydrogenase.

The binary complex of NAD+ and dogfish A4 lactate dehydrogenase reacts reversibly with pyruvate enol to produce an inactive, enzyme-adduct complex, in which the nicotinamide and pyruvate moieties are linked by means of a covalent bond. This process is examined in both the forward and reverse directions as a function of reactant and buffer concentrations at pH 7, under conditions where the enolization of pyruvate is at equilibrium, and the involvement of complexes with stoichiometry E.NAD, E.NAD.PyrE, and E.NAD.PyrK is defined. (The subscripts, E and K, indicate the enol and keto forms of pyruvate.) One pathway for formation of the adduct complex involves the prior formation of the E.NAD.PyrE complex from E.NAD and pyruvate enol; the alternative pathway involves formation of the same complex via the enolization of E.NAD.PyrK, a process that is catalyzed by an external (nonenzymic) base. The possible use of the adduct reaction as a model for the normal enzymic reaction is considered.

Animals↗

PubMed1984

Purification of lactate dehydrogenase isoenzymes one, two, and three from human erythrocytes.

Lactate dehydrogenase (LD) isoenzymes 1, 2, and 3 were prepared from human erythrocytes by sequential ion-exchange chromatography followed by general-ligand (AMP analog) affinity chromatography. Respective yields, purification factors, and specific activities (kU per gram of protein) were 25%, 4394-fold, and 209.7; 40% 4385-fold, and 199.1; and 18%, 7565-fold, and 192.9. The respective preparations contained less than 0.5% of contaminating LD isoenzyme activity as judged from electrophoresis on thin-layer agarose, were homogeneous as judged by electrophoresis on polyacrylamide gel (both in the presence and absence of sodium lauryl sulfate), and showed minor contamination by other LD isoenzymes as judged by analytical isoelectric focusing. We think that these preparations would be useful as human-based calibrating or reference materials. Their purity is such that these preparations could also be used as antigens for the development of suitable antisera.

Chromatography, Affinity↗

PubMed1979

Lifetimes and NADH quenching of tryptophan fluorescence in pig heart lactate dehydrogenase.

The decay of tryptophan emission from pig heart lactate dehydrogenase following pulsed excitation has been recorded in Tris buffer solution at pH 7.4. All tryptophan residues emit. A good least-squares two-component fit is obtained with I(t)-0.53e-t/1.2 + 0.47e-t/68. A longer lived emitter (r=7.4--8.1 ns) is also observed. Bound NADH strongly quenches most of the 6.8-ns emission, but the 1.2-ns component is relatively unaffected. The fluorescence is moderately quenched by acrylamide and only slighty quenched by I- and Cs+. The pulsed and steady-state fluorescence is discussed in terms of a model with three lifetime classes of tryptophan, viz., 1, 4, and 8 ns. The three-dimensional structure of the enzyme--NADH complex is used to develop a description of the individual residues in terms of their lifetimes and sensitivity to NADH and I- quenching. The nonlinear NADH quenching is due to intersubunit energy transfer from Trp-248 to NADH.

Animals↗

PubMed1978

Lactate dehydrogenase in estrogen-responsive human breast cancer cells.

Lactate dehydrogenase activity (LDH) was measured in the MCF-7 human breast cancer cell line derived at the Michigan Cancer Foundation from a patient with metastatic breast adenocarcinoma. LDH was found in the 46,000 X g supernatant of cell lysates, but not in the culture medium. Only the fifth isozyme (LDH-5) could be demonstrated by cellulose acetate electrophoresis and relative heat inactivation studies. When endogenous steroids were removed from the medium, addition of estrogen to the growth medium for several days elevated LDH 2-fold above controls; LDH was not altered when MCF-7 cells were treated with progesterone, hydrocortisone, prolactin, insulin, or triiodothyronine. A physiological concentration (0.1 nM) of 17beta-estradiol was sufficient to produce a maximal LDH increase. There were no qualitative isozyme changes in response to estrogen. LDH activity may therefore be a useful marker protein for studying hormone action in the MCF-7 human breast cancer cell line.

Adenocarcinoma↗

PubMed1978

[Changes in lactate dehydrogenase isoforms in the process of oncogenesis].

Isoenzymes of lactate dehydrogenase were studied by disc-electrophoresis in polyacrylamide gel, and in the clinic--in 1% agar gel. Oncovirus A12 invasion of the culture of rat embryo fibroblasts (REF) was found to result in the increased percentage of the cathode fractions activity (LDG-4 and LD-5) and in the disappearance of LDG-1 yet during the first day of the experiment prior to hypoxia and enhanced proliferation, i. e. it is most likely to be primary. In the homogenates of cancerous tumor and large intestine polyps of man also a reliable increase of the cathode and a decrease or disappearance of the anode fractions accur. A correlation of the experimental and clinical data allowed a suggestion to be made that LDG isoenzymes changes are genetically conditioned and play an important role in the process of oncogenesis, providing conditions for the increased intensity of glycolysis and proliferation.

Adenoviruses, Human↗

PubMed2002

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 7. Certification of four reference materials for the determination of enzymatic activity of gamma-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase accord.

This paper is the seventh in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of Gamma-Glutamyltransferase. A document describing the determination of preliminary reference values is also in preparation. The certification of the catalytic activity concentrations as determined by the recently elaborated IFCC primary reference methods at 37 degrees C of four enzyme preparations, namely IRMM/IFCC 452 (gamma-glutamyltransferase), IRMM/IFCC 453 (lactate dehydrogenase 1), IRMM/IFCC 454 (alanine aminotransferase) and IRMM/IFCC 455 (creatine kinase) is described. Homogeneity data were derived from previous results. Stability was assessed using recently obtained data as well as data from previous stability studies. The collaborative study for value assignment was performed under a strict quality control scheme to ensure traceability to the primary reference method. Uncertainty of the materials was assessed in compliance with the Guide to the Expression of Uncertainty in Measurement. The certified values obtained at 37 degrees C are 1.90 microkat/l +/- 0.04 microkat/l (114.1 U/l +/- 2.4 U/l), for gamma-glutamyltransferase, 8.37 microkat/l +/- 0.12 microkat/l (502 U/l +/- 7 U/l), for lactate dehydrogenase 1, 3.09 microkat/l +/- 0.07 microkat/l (186 U/l +/- 4 U/l), for alanine aminotransferase and 1.68 microkat/l +/- 0.07 microkat/l (101 U/l +/- 4 U/l), for creatine kinase. The materials are intended for internal quality control as well as for the evaluation of test systems as required by recent European Union legislation. Furthermore, the materials can be used to transfer accuracy from a reference method to a routine procedure provided the procedures exhibit the same analytical specificity and the certified materials are commutable.

Alanine Transaminase↗

PubMed2001

A novel missense mutation in human lactate dehydrogenase B-subunit gene.

Reduced activity of serum lactate dehydrogenase (LDH; EC 1.1.1.27) was found in a male medical student during practical examinations of his own blood. Serum LDH isoenzyme pattern showed reductions in activities of the isoenzymes with lower subunit A/B ratios such as LDH1 and LDH2. These findings were indicative of a partial LDH-B subunit deficiency, which was confirmed in erythrocyte hemolysates by Western blotting. Polymerase chain reaction (PCR)-based DNA sequence analysis of the LDH-B subunit gene revealed a heterozygous nucleotide change: a guanine to adenine substitution in codon 69 (GGG --> GAG) at the third exon of the LDH-B subunit gene that resulted in a glycine to glutamic acid substitution (G69E). The mutation was confirmed by PCR-restriction fragment length polymorphism (RFLP) analysis using a mismatched primer to introduce a new NcoI restriction site. The same heterozygous mutation was found in his mother but not in other family members. This mutation involves a residue belonging to alphaC helix in LDH-B subunit protein molecule that functions as an interface for other subunits.

Adult↗

PubMedDate not supplied

Chemosensitivity testing of fresh and continuous tumor cell cultures using lactate dehydrogenase.

We have examined the use of the LDH (lactate dehydrogenase) assay for chemosensitivity testing in established and primary cultures of sarcoma, leukaemia and ovarian cancer in parallel with the MTT assay. The method we describe is rapid, sensitive and ideal for 96-well plate assays using adherent or suspension cultures. Excellent agreement between the two methods was observed (r = 0.936) using a variety of antitumour agents, with some notable exceptions. In the Bax (human synovial sarcoma) cell line MTT colour production by control cells was very low, thus MTT-->formazan production could not be relied upon as a definitive end point equating with cell number. In contrast, colour production of control cells using the LDH assay was significantly greater and all cultures tested were suitable for titration of chemosensitivity. There was a discrepancy between IC50 values obtained either by cell counting or MTT in the HTB88 (human leiomyosarcoma) line treated with 5-FU (59.9 microM vs > 200 microM, respectively). However, cell counting agreed well with the LDH assay (IC50 47.3 microM). Whilst the MTT assay remains a reliable method for chemosensitivity testing, the LDH assay may prove more appropriate in certain experimental settings.

Cell Survival↗

PubMed1994

Integration of aqueous two-phase extraction and affinity precipitation for the purification of lactate dehydrogenase.

Integration of extraction in aqueous two-phase system and affinity precipitation was investigated as a technique for purification of lactate dehydrogenase (LDH) from porcine muscle extract. An enteric coating polymer, Eudragit S 100, which can be made reversibly soluble and insoluble by change in pH was used as the ligand carrier. The ligand used was Cibacron blue 3GA. The polymer is nearly totally partitioned to the top phase (> 98%) in PEG-dextran aqueous two-phase system. The enzyme, lactate dehydrogenase, was first spontaneously partitioned to the bottom phase in a 6% (w/w) PEG 8000-8% (w/w) dextran T250 phase system. New PEG phase and Eudragit-dye were then added to the bottom phase, which helped in extraction of LDH to the top phase. After a washing step with a fresh bottom phase, Eudragit-dye-target protein affinity complex was precipitated out from the top phase by lowering the pH to 5.1. The enzyme was recovered by treatment of the complex with 0.5 M NaCl with a yield of 54% and a specific activity of 245 units/mg. The purification of LDH by this procedure was better than that obtained by a single step of affinity partitioning.

Acrylic Resins↗

PubMed1987

Lactate dehydrogenase isoenzymes in serum during recent acute myocardial infarction.

Lactate dehydrogenase (LD, EC 1.1.1.27) isoenzymes 1 and 2 and the LD 1:2 ratio were determined in 62 patients with recent myocardial infarction 24, 48, and 72 h after total serum LD activity had returned to normal values. From the results we could define two groups of patients. The first, 40 patients in whom proportions of LD-1 and LD-2 isoenzymes in serum and the LD 1:2 ratio were all within the normal reference interval, all had an uncomplicated course of recovery from myocardial infarction. In the remaining 22 patients, LD-1 still exceeded LD-2 24 to 72 h after total LD activity returned to normal values; i.e., the ratio was similar to that in patients with myocardial infarction. Seven of these 22 patients (32%) had a complicated course, with re-infarction in all seven. Thus, even in the presence of normal total LD activity, a high LD 1:2 ratio may reflect a consistent focal myocardial necrosis in some patients with recent myocardial infarction and may serve as an early marker for further re-infarction.

Adult↗

PubMed1979

[Liver tissue lactate dehydrogenase isoenzymes in cirrhosis].

Isoenzymes of the liver tissue lactate dehydrogenase (LDG) were examined in 37 patients with micro- and macronodular cirrhosis. LDG isoenzymes were studied by histochemical and cytophotometrical methods, electrophoresis in polyacrylamide gel, densitometry. The results were treated statistically. Uneven (mosaic) distribution of LDG activity in hepatocytes was observed in hepatocirrhosis. Different hepatocytes and fibroblasts had dissimilar sets of LDG isoenzymes. Anodal isoenzymes showed a higher activity in the bile duct epithelium. Cathodal isoenzymes in hepatocytes were localized mostly near the external cell membrane and the nuclear membrane. Micronodular cirrhoses are characterized by an increased activity of LDG2 and LDG3 and a decreased activity of LDG5 in the liver tissue. Macronodular cirrhoses are characterized by a decreased activity of LDG1 and LDG3 and an increased activity of LDG5. In the presence of hepatocyte insufficiency the activity of LDG1 decreased. Changes in the liver tissue LDG isoenzymes profile is due to a number of degenerative and regeneratory lesions typical of the cirrhotic process. The determination of the profile of the liver tissue LDG isoenzymes is important for the establishment of the type of cirrhosis, the degree of hypoxia, the intensity of regeneration processes, and the effectiveness of the therapy given to the patient.

Autopsy↗

PubMed1984

Effect of hyperthermia and gamma-radiation on Escherichia coli K1060 D-lactate dehydrogenase.

The response of E. coli K1060 D-lactate dehydrogenase (D-LDH), an enzyme located in the cytoplasmic membrane, was studied following 42.5 degrees C hyperthermia and/or gamma-irradiation. The inactivation of D-LDH following the above treatment was used as a tool to probe the role of membrane proteins in the radiation and/or heat sensitivity of cells. No correlation between loss of enzyme activity and cell killing was found, suggesting that D-LDH does not play an important role in hyperthermic cell survival. The results obtained in combined hyperthermia and gamma-irradiation treatments on loss of D-LDH activity and E. coli cell killing suggest that an interaction between heat and radiation occurs at the membrane structure level. Moreover, when cells were heated at 42.5 degrees C in the presence of 10 mM procaine-HCl, both cell killing and loss of D-LDH activity were enhanced. The involvement of membrane structure in the heat sensitivity of cells is strongly indicated by the latter observations. The opposite effect was observed when procaine was present during irradiation in oxic conditions, suggesting that procaine itself can also act as a scavenger towards OH-induced radicals.

Cell Survival↗

PubMed1994

Cloning, characterization and insertional inactivation of the Lactobacillus helveticus D(-) lactate dehydrogenase gene.

A plasmid, designated pSUW100, encoding the D(-)lactate dehydrogenase [D(-)-LDH; NAD+ oxidoreductase, EC 1.1.1.28] from Lactobacillus helveticus CNRZ32 was identified from a genomic library by complementation of Escherichia coli FMJ39. The D(-)LDH gene was localized by Tn5 mutagenesis and subcloning to a 1.4-kb region of pSUW100. A 2-kb DraI fragment of pSUW100 encoding D(-)LDH activity was subcloned and its nucleotide sequence determined. Analysis of this sequence identified a putative 1,014-bp D(-)LDH open reading frame that encodes a polypeptide of 337 amino acid residues with a deduced molecular mass of 38 kDa. The distribution of homology to the CNRZ32 D(-)LDH gene in several lactic acid bacteria was determined by Southern hybridization using an internal fragment of the D(-)LDH gene as a probe. Hybridization was detected in leuconostocs and pediococci but not in lactococci or Lactobacillus casei. An integration plasmid was constructed from pSA3 and a 0.60-kb internal fragment of the D(-)LDH gene. This plasmid was used to construct a D(-)LDH-negative derivative of L. helveticus CNRZ 32 by gene disruption; this derivative was determined as producing only L(+)lactic acid. No significant difference in growth or total lactic acid production was observed between CNRZ32 and its D(-)LDH mutant.

Amino Acid Sequence↗

PubMed1992

[Interaction of lactate dehydrogenase and the sarcoplasmic reticulum membrane].

The binding of lactate dehydrogenase (LDH) to sarcoplasmic reticulum membranes results in a 60-70% decrease of the enzyme specific activity. This binding occurs both in high (Kd = 1 microM) and low affinity sites. Addition of NADH or NAD+ and a increase of ionic strength lead to the solubilization of the bound enzyme. A similar effect is observed after addition of the fluorescent probes--anilinonaphthalene sulfonate (ANS) and auramine O (A0). The effect of ANS consists predominantly in its binding to the membrane, while that of A0 is due to the probe interaction with the enzyme. At low concentrations of toluidinylnaphthalene sulfonate (TNS) under conditions of predominant binding of the probe to the membrane, the LDH binding to microsomes is enhanced. A rise in the TNS concentration leads to the formation of the probe-LDH complex which interaction with membrane is hampered. The sites of the probes binding to the protein are located outside the enzyme active center but are, nevertheless, sensitive to it states. It is assumed that these sites of the LDH molecule are involved in its interaction with the membrane. The decline of activity of the bound enzyme is interpreted in terms of alterations of the physico-chemical properties of the medium during the enzyme transition from the solution to the perimembrane space.

Anilino Naphthalenesulfonates↗

PubMed1992

Unusual amino acid substitution in the anion-binding site of Lactobacillus plantarum non-allosteric L-lactate dehydrogenase.

In Lactobacillus plantarum non-allosteric L-lactate dehydrogenase (L-LDH), the highly conserved His188 residue, which is involved in the binding of an allosteric effector, fructose 1,6-bisphosphate [Fru(1,6)P2], in allosteric L-LDH is uniquely substituted by an Asp. The mutant L. plantarum L-LDH, in which Asp188 is replaced by a His, showed essentially the same Fru(1,6)P2-independent catalytic activity as the wild-type enzyme, except that the Km and Vmax values were slightly decreased. However, the addition of Fru(1,6)P2 induced significant thermostabilization of the mutant enzyme, as in the case of many allosteric L-LDHs, while Fru(1,6)P2 showed no significant effect on the stability of the wild-type enzyme, indicating that only the single-point mutation, G-->C, sufficiently induces the Fru(1,6)P2-binding ability of L. plantarum L-LDH. The mutant enzyme showed higher thermostability than the wild-type enzyme in the presence of Fru(1,6)P2. In the absence of Fru(1,6)P2, on the other hand, the mutant enzyme was more labile below 65 degrees C but more stable above 70 degrees C.

Allosteric Regulation↗

PubMed1976

Properties of the testicular lactate dehydrogenase isoenzyme.

1. Studies were carried out with pure lactate dehydrogenase isoenzymes C4 (LDH isoenzyme X), B4, (LDH isoenzyme 1) and A4 (LDH isoenzyme 5) isolated from mouse testis, heart and muscle tissue respectively; with LDH isoenzyme X purified from pigeon testes and with crude lysates of spermatozoa from man, bull and rabbit. 2. LDH isoenzyme X from all species showed greater ability than the other isoenzymes to catalyse the NAD+-linked interconversions of 2-oxobutanoate into 2-hydroxybutanoate and of 2-oxopentanoate into 2-hydroxypentanoate. 3. Mouse LDH isoenzyme X presented the broadest spectrum of substrate specificity. It exhibited very similar Km values for a variety of 2-oxo acids: 2-oxopropanoate (pyruvate), 2-oxobutanoate, 2-oxo-3-methylbutanoate, 2-oxopentanoate, 2-oxo-3-methylpentanoate, 2-oxo-4-methylpentanoate, 2-oxohexanoate and 2-oxo-3-phenylpropanoate (phenylpyruvate). The corresponding 2-hydroxy acids were also readily utilized in the reverse reaction. A strong inhibition by substrate and product was demonstrated for the direct reaction. 4. Intracellular distribution of LDH isoenzyme X was investigated in mouse testes. LDH isoenzyme X activity was located in the fraction of "heavy mitochondria" and in the soluble phase. 5. A possible functional role for LDH isoenzyme X is proposed: the redox couple-2-oxo acid-2-hydroxy acid could integrate a shuttle system transferring reducing equivalents from cytoplasm to mitochondria.

Animals↗