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PubMed1990

Significance of elevated serum LDH (lactate dehydrogenase) activity in atopic dermatitis.

Serum lactate dehydrogenase activity (LDH) was elevated in most cases with the severe type of atopic dermatitis (AD). We examined whether LDH correlated specifically with the clinical courses and the severity of AD skin eruptions. Blood eosinophil numbers (Eo), LDH and its isoenzymes, and serum IgE (IgE) levels in eighty patients with AD were measured before and after treatment. In improved groups, Eo and LDH decreased or returned to normal values after treatment. In contrast, both levels increased in patients who worsened after treatment. Thus changes in Eo and LDH correlated well with the clinical picture. However, IgE levels did not correlate with the clinical condition. High LDH levels were detected in the severe type of AD more frequently than in the mild type. A large majority of patients with elevated LDH (higher than 300 IU/l) before treatment had more severe skin eruptions. When the LDH before treatment was within normal limits, the LDH levels in improved patients showed a further decrease. Eo, LDH and IgE were correlated closely with the clinical scores of AD, and the relationship between LDH and clinical score was the highest. Elevations of LDH5 and LDH4 among the LDH isoenzymes were more prominent in the severe type of AD than in the mild type. We concluded that LDH was useful as a marker for evaluating the disease condition and severity of skin eruption of AD.

Adolescent↗
PubMed1983

Lactate dehydrogenase isoenzymes in hamster lung lavage fluid after lung injury.

Lactate Dehydrogenase Isoenzymes in Hamster Lung Lavage Fluid after Lung Injury. Beck, B. D., Gerson, B., Feldman, H. A., and Brain, J. D. (1983). Toxicol. Appl. Pharmacol. 71, 59-71. Lactate dehydrogenase (LD) levels and isoenzyme patterns were determined in the cell-free supernatant fractions of lung lavage fluid from hamsters exposed to alpha-quartz, iron oxide, Triton X-100, 100% O2, or 200 ppm SO2. The isoenzyme patterns were compared to those derived from hamster lung homogenates, serum, polymorphonuclear neutrophils (PMNs), pulmonary macrophages, and red blood cells. The isoenzyme patterns from alpha-quartz- and iron oxide-exposed animals resembled each other and were similar to that of PMNs. In contrast, the pattern seen after Triton X-100 exposure was similar to those of whole lung homogenates and of red blood cells. A 96-hr exposure to 100% O2 yielded an LD isoenzyme pattern in lung lavage fluid similar to that of serum. Exposure to SO2 did not alter LD levels, showing that upper airways damage is not reflected by changes in LD in lung lavage fluid. We conclude that LD isoenzyme patterns of lung lavage fluid can be used to differentiate among types of pulmonary injury and may help identify the sites of injury.

Animals↗
PubMed1992

Evidence for temperature-dependent conformational changes in the L-lactate dehydrogenase from Bacillus stearothermophilus.

L-Lactate dehydrogenase from Bacillus stearothermophilus (BSLDH) has been shown to change its conformation in a temperature-dependent manner in the temperature range between 25 and 70 degrees C. To provide a more detailed understanding of this reversible structural reorganization of the tetrameric form of BSLDH, we have determined in the presence of 5 mM fructose, 1,6-bisphosphate (FBP) the effect of temperature on far-UV and near-UV circular dichroism (CD), Nile red-binding to the enzyme surface, NADH binding, fluorescence polarization of fluorescamine-labeled protein, and hydrogen-deuterium exchange. In addition, we have analyzed the temperature dependence of the dimer-tetramer equilibrium of this protein by steady-state enzyme kinetics in the absence of FBP. The results obtained from these measurements at various temperatures can be summarized as follows. No changes in the secondary-structure distribution are detectable from far-UV CD measurements. On the other hand, near-UV CD data reveal that changes in the arrangements of aromatic side chains do occur. With increasing temperature, the asymmetry of the environment around aromatic residues decreases with a small change at 45 degrees C and a more pronounced change at 65 degrees C. Nile red-binding data suggest that the BSLDH surface hydrophobicity changes with temperature. It appears that decreasing the surface hydrophobicity may be a strategy to increase the protein stability of the active enzyme. We have noted significant alterations in the thermodynamic binding parameters of NADH above 45 degrees C, indicating a conformational change in the active site at 45 degrees C. The hydrodynamic volume of BSLDH is also temperature dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1982

Studies of lactate dehydrogenase in the purified state and in intact erythrocytes.

Lactate dehydrogenase in intact erythrocytes was studied by observing isotope exchange between lactate and pyruvate by p.m.r. The inhibition of the enzyme in intact cells by both oxalate and pyruvate was found to be similar to that of the purified enzyme. The activity of the enzyme in intact cells indicates that the free solution NAD+ + NADH concentration in erythrocytes is about 10 microM whereas the total extractable NAD+ + NADH is about 80 nmol/ml of cell water.

Erythrocytes↗
PubMed1989

Characterization and comparison of epsilon-crystallin and lactate dehydrogenases in the lenses of vertebrates and invertebrates.

Screening of lens homogenates for the identification of lactate dehydrogenases was undertaken for the representative species from five major classes of vertebrates plus the cephalopod of invertebrates. The duck and caiman lenses appeared to contain the highest enzymatic activity of this glycolytic enzyme among all species examined. Biochemical isolation and characterization of epsilon-crystallins from the duck and caiman lenses revealed differences between these structural crystallins and the authentic lactate dehydrogenase of the avian heart regarding some of the kinetic properties. This is in contrast with the claim that duck epsilon-crystallin is identical to heart-type lactate dehydrogenase.

Amino Acids↗
PubMed1997

Evaluation of the role of lactate dehydrogenase in oxalate synthesis.

The kinetic properties of lactate dehydrogenase (LDH) (EC 1.1.1.27) from spinach leaves were studied in order to evaluate the possible roles of LDH in the production of oxalate. LDH was purified by affinity chromatography on affigel blue and oxamate agarose columns. The pH optimum for the reduction of pyruvate was 7.25, while that for the reduction of glyoxylate was 7. The rate of reduction of pyruvate and glyoxylate at the optimum pH indicated substrate inhibition at concentrations above 8 and 40 mM, respectively. The maximum activity of LDH with pyruvate was about three times greater than with glyoxylate. The pH optimum for the oxidation of lactate was 9, with very low activity below pH 8. Substrate inhibition was apparent at lactate concentrations above 10 mM. LDH was inactive with glyoxylate in the oxidative reaction, which would lead to the biosynthesis of oxalate.

Catalysis↗
PubMed2004

Observation and identification of lactate dehydrogenase anomaly in a postburn patient.

OBJECTIVE: Lactate dehydrogenase (LDH) anomaly is one of the macroenzymes. Macroenzymes are enzymes in serum that have formed high molecular mass complexes, either by self polymerisation or by association with other serum components. The aim of this study was to identify the properties of LDH anomaly and observe the changes from admission to discharge in a postburn patient with LDH anomaly in his serum. METHODS: LDH isoenzymes of the serum were electrophoretically fractionated with terylene cellulose acetate supporting media; LDH anomaly was identified by counter immunoelectrophoresis. RESULTS: An abnormal LDH-4 band and an extra band on the cathode of LDH-5 were observed in the serum of this patient and were found to be part of an LDH-IgG complex. As his symptoms improved, the patient's LDH anomaly gradually disappeared. The appearance and disappearance of the anomaly seemed to be related to the progression of the patient's burns. CONCLUSION: In clinical practice, it is important to keep in mind the possibility of an LDH anomaly in patients when the LDH level is abnormally high or does not seem to be related to the clinical state. Early discovery of an LDH anomaly in a patient's serum may be useful for diagnosis and treatment.

Burns↗
PubMed1992

Construction of a stable dimer of Bacillus stearothermophilus lactate dehydrogenase.

A molecular graphics analysis of the features which prevent cytosolic malate dehydrogenase dimers from forming tetramers was evaluated by its success in predicting the synthesis of a version of the LDH framework which is a stable dimer. Surface residues responsible for malate dehydrogenases being dimers were revealed by superimposing the structures of two dimers of pig cytosolic malate dehydrogenase on one homologous tetramer of L-lactate dehydrogenase from Bacillus stearothermophilus. Four regions were identified as composing the P-axis dimer-dimer interface. Two regions of the dimer were surface loops that collided when built as a tetramer: a large loop (residues 203-207, KNOBI) and a small loop (residues 264-269, KNOBII), and these were candidates to explain the dimeric character of malate dehydrogenase. The analysis was tested by constructing a synthetic B. stearothermophilus lactate dehydrogenase (KNOBI) containing the large malate dehydrogenase loop (residues 203-207 being AYIKLQAKE, and extra four amino acids). The new construct was thermotolerant (90 degrees C) and enzymically active with kcat and KM (pyruvate) values similar to those of the wild-type enzyme. However, whereas the allosteric activator fructose 1,6-bisphosphate decreased KM 100 times for wild type, it had no influence on KNOBI. The molecular volumes of 1-120 microM concentrations of the construct were measured by time-resolved decay of tryptophan fluorescence anisotropy and by gel filtration. Both methods showed the molecular weight of wild type increased from dimer to tetramer with Kd about 20 microM dimer. KNOBI remained a dimer under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1982

Lactate dehydrogenase in Phycomyces blakesleeanus.

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.

Adenosine Triphosphate↗
PubMed2003

Mid-trimester amniotic fluid angiogenin, lactate dehydrogenase and fibronectin in the prediction of preterm delivery.

OBJECTIVE: Our purpose was to compare angiogenin, lactate dehydrogenase (LDH) and fibronectin levels in mid-trimester amniotic fluid of patients with preterm and term deliveries and to find out their predictive values for preterm birth. STUDY DESIGN: A prospective cohort study was conducted in 55 pregnancies with singleton gestations that underwent amniocentesis at 15-20 weeks for standard genetic indications. Amniotic fluid angiogenin, lactate dehydrogenase and fibronectin levels were measured by enzyme-linked immunosorbent assay (ELISA), radial immundiffusion technic and automated analyzer, respectively. RESULTS: Five patients delivered preterm, five developed signs or symptoms of threatened preterm labor and 45 had term delivery after an uneventful pregnancy. Demographic data were not significantly different. Amniotic fluid angiogenin and lactate dehydrogenase levels were significantly higher in patients with preterm than term deliveries (P<0.001 and 0.02, respectively). Receiver-operator characteristic curve analysis showed that the amniotic fluid angiogenin had the best screening efficiency in predicting preterm delivery. An angiogenin level of 35ng/ml was the optimal cut-off value for the prediction of preterm delivery, with a sensitivity of 100% and specificity of 91%. CONCLUSION: Second-trimester angiogenin is found to be quite effective in the prediction of preterm delivery. Preexisting intrauterine ischemia may be an important risk factor for preterm delivery and already be present in the early mid-trimester.

Adult↗
PubMed1979

Purification and properties of the flavoenzyme D-lactate dehydrogenase from Megasphaera elsdenii.

A pyridine nucleotide independent D-lactate dehydrogenase has been purified to apparent homogeneity from the anaerobic bacterium Megasphaera elsdenii. The enzyme has a molecular weight of 105 000 by sedimentation equilibrium analysis with a subunit molecular weight of 55 000 by sodium dodecyl sulfate gel electrophoresis and is thus probably a dimer of identical subunits. It contains approximately 1 mol of FAD and 1 g-atom of Zn2+ per mol of protein subunit, and the flavin exhibits a fluorescence 1.7 times that of free FAD. An earlier purification [Brockman, H. L., & Wood, W. A. (1975 J. Bacteriol. 124, 1454--1461] results in substantial loss of the enzyme's zinc, which is required for catalytic activity. The new purification yields greater than 5 times the amount of enzyme previously isolated. The enzyme is specific for D-lactate, and no inhibition is observed with L-lactate. Surprisingly, the enzyme has a significant oxidase activity, which depends on the ionic strength. Vmax values of 190 and 530 min-1 were obtained at a gamma/2 of 0.224 and 0.442, respectively. Except for this atypically high oxygen reactivity, D-lactate dehydrogenase resembles other flavoenzyme dehydrogenases in that the flavin does not react with sulfite, the tryptophan content is low, and a neutral blue semiquinone is formed upon photochemical reduction. The enzyme flavin is reduced either by dithionite, by oxalate plus catalytic 5-deazaflavin in the presence of light, or by D-lactate. Two electrons per flavin were consumed in a dithionite titration, implyine with varying ratios of D-lactate and pyruvate, an Em7 of -0.219 +/- 0.007 V at 20 degrees C was calculated for the flavin. The enzyme requires dithiothreitol for stability. Rapid inactivation results when the enzyme is incubated with a substoichiometric level of Cu2+. This inactivation can be reversed by dithiothreitol. It is proposed that the enzyme possesses a pair of cysteine residues capable of facile disulfide formation.

Amino Acids↗
PubMed1977

Lactate dehydrogenase inhibitors in NADH preparations.

The presence of a new lactate dehydrogenase inhibitor on the trailing edge of the NADH peak from chromatography on diethylaminoethyl-celluose [Loshon et al., Clin. Chem., this issue] was verified. It was resolved from the NADH by high-performance liquid chromatography on muBondapak C18. When the new inhibitor was present in a reaction mixture to the extent that, of the initial 260-nm absorbance, about 5% was contributed by the inhibitor, the rate of NADH oxidation by lactate dehydrogenase decreased by 65%. The inhibitor absorbs at 260 and 340 nm, and is different from the Strandjord-Clayson inhibitor [J. Lab. Clin. Med. 67, 144 (1966)] by both types of chromatography. Because this new inhibitor has ultraviolet properties similar to those of NADH and chromatographs with the NADH on DEAE-cellulose, the high-performance liquid chromatographic method must be used to ensure its absence in preparations of NADH used for clinical assay.

Chromatography, DEAE-Cellulose↗
PubMed1996

Relevance of lactate dehydrogenase activity to the control of oxidative glycolysis in pancreatic islet B-cells.

The activities of hexokinase isoenzymes, lactate dehydrogenase, cytosolic NAD-linked glycerophosphate dehydrogenase, mitochondrial FAD-linked glycerophosphate dehydrogenase, and glutamate dehydrogenase were measured in homogenates of rat purified pancreatic B and non-B islet cells. In B cell homogenates, the maximal activity of hexokinase and glucokinase was one to two orders of magnitude lower than that of lactate dehydrogenase. The activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase was also much lower than that of the cytosolic NAD-linked glycerophosphate dehydrogenase . A comparable hierarchy in the activity of these enzymes was observed in non-B islet cells. These findings reinforce the view that the preferential stimulation of oxidative glycolysis observed in insulin-producing cells, when exposed to high concentrations of D-glucose, is attributable to a Ca2+-induced activation of the mitochondrial FAD-linked glycerophosphate dehydrogenase, rather than to saturation of the catalytic activity of lactate dehydrogenase.

Animals↗
PubMed1990

Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui.

D-Lactate dehydrogenase from the extreme halophilic archaebacterium Halobacterium marismortui has been partially purified by ammonium-sulfate fractionation, hydrophobic and ion exchange chromatography. Catalytic activity of the enzyme requires salt concentrations beyond 1M NaCl: optimum conditions are 4M NaCl or KCl, pH 6-8, 50 degrees C. Michaelis constants for NADH and pyruvate under optimum conditions of enzymatic activity are 0.070 and 4.5mM, respectively. As for other bacterial D-specific lactate dehydrogenases, fructose 1,6-bisphosphate and divalent cations (Mg2+, Mn2+) do not affect the catalytic activity of the enzyme. As shown by gel-filtration and ultracentrifugal analysis, the enzyme under the conditions of the enzyme assay is a dimer with a subunit molecular mass close to 36 kDa. At low salt concentrations (less than 1M), as well as high concentrations of chaotropic solvent components and low pH, the enzyme undergoes reversible deactivation, dissociation and denaturation. The temperature dependence of the enzymatic activity shows non-linear Arrhenius behavior with activation energies of the order of 90 and 25 kJ/mol at temperatures below and beyond ca. 30 degrees C. In the presence of high salt, the enzyme exhibits exceptional thermal stability; denaturation only occurs at temperatures beyond 55 degrees C. The half-time of deactivation at 70 and 75 degrees C is 300 and 15 min, respectively. Maximum stability is observed at pH 7.5-9.0.

Archaea↗
PubMed1989

Thermodynamic nonideality in enzyme catalysis. Effect of albumin on the reduction of pyruvate by lactate dehydrogenase.

The enhanced catalytic reduction of pyruvate by rabbit muscle lactate dehydrogenase that results from the addition of serum albumin [Nichol, L. W., Sculley, M. J., Ward, L. D. & Winzor, D. J. (1983) Arch. Biochem. Biophys. 222, 574-581] is shown to emanate solely from an increase in maximal velocity, there being no discernible effect of this inert space-filling macromolecular solute on the Michaelis constant for either pyruvate or NADH. As part of the search for a mechanistic explanation of this kinetic phenomenon, the space-filling effects of albumin have been used to eliminate the possibility that the increase in sedimentation coefficient of lactate dehydrogenase effected by inclusion of oxamate with enzyme-NADH complex reflects preferential binding of this pyruvate analog to a more compact isomeric state of the binary complex. The enzyme kinetic results are therefore considered in terms of a reaction scheme entailing gross conformational changes during the formation of ternary enzyme-NADH-pyruvate complex and its isomerization to an activated transition state. The experimentally observed insensitivity of the Michaelis constant for pyruvate to albumin concentration is in keeping with theoretical prediction, but incorporation of the measured extent of maximal velocity enhancement into the kinetic model leads to a predicted volume for the fully saturated transition-state complex that is far too small to be experimentally feasible. A more complex mechanistic model involving additional isomerizations of enzyme-substrate species is thus required to achieve quantitative description of the albumin effect solely in terms of thermodynamic nonideality.

Kinetics↗
PubMed1980

Improved serum-initiated aspartate aminotransferase assay by inhibition of lactate dehydrogenase with oxamate.

We describe a serum-inititated aspartate aminotransferase (EC 2.6.1.1) assay that obviates the need for added lactate dehydrogenase (EC 1.1.1.27) in the reagent system. Interference from the oxidation of NADH by endogeneous lactate dehydrogenase is eliminated by adding sodium oxamate, a competitive inhibitor of the enzyme. The advantages of oxamate inhibition over lactate dehydrogenase addition are a shorter preincubation period, an increase in the linear range from less than 600 to more than 1400 U/L, no interference from above-normal concentrations of ketoacids, and elimination of bias between serum-initiated and the standard oxoglutarate-initiated assays for aspartate aminotransferase.

Amino Acids↗
PubMed1986

13C-NMR and transient kinetic studies on lactate dehydrogenase [Cys(13CN)165]. Direct measurement of a rate-limiting rearrangement in protein structure.

Chemical modification of cysteine-165 in pig heart lactate dehydrogenase to produce lactate dehydrogenase [Cys(13CN)165] introduces an covalently bound, enriched 13C probe at a position adjacent to the active cen. The signal from the thiocyanate probe is clearly visible at 47 ppm relative to dioxane. On formation of binary complexes with NAD+ and NADH, no signal change is detected. Formation of the ternary complexes E-NADH-oxamate and E-NAD+-oxalate results in an upfield shift of the signal of 1.2 ppm. These results interpreted as demonstrating that binding of the substrate analogue induces a conformational change a position adjacent to the active centre. Exchange experiments in which the enzyme is poised in dynamic equilibrium between binary and ternary complexes show that the rate at which the probe senses a change environment is the same as the kinetically observed unimolecular event which limits the enzyme-catalyst reduction of pyruvate. The two processes show the same dependence on temperature, solvent composition and pH. These results indicate that the rate-limiting isomerisation corresponds to a rearrangement of the protein in the region of cysteine-165.

Amino Acid Sequence↗
PubMed1996

The discriminating value of serum lactate dehydrogenase levels in children with malignant neoplasms presenting as joint pain.

OBJECTIVES: To determine if serum lactate dehydrogenase levels distinguish patients with malignant neoplasm presenting with musculoskeletal complaints from patients with juvenile rheumatoid arthritis who reported similar symptoms. DESIGN: Retrospective case-comparison study. SETTING: Tertiary care, outpatient clinics. PATIENTS: Twelve patients with malignant neoplasms who presented with arthritis or arthralgias and normal complete blood cell counts and blood smears in whom rheumatologic diagnosis was initially made were compared with 24 children with a final diagnosis of juvenile rheumatoid arthritis. The patients with malignant neoplasms all had normal blood counts and elevated sedimentation rates at symptom onset. INTERVENTIONS: None. RESULTS: Serum lactate dehydrogenase levels were significantly higher in the cancer patients at 2.2 times the normal values vs 0.8 times high normal for patients with juvenile rheumatoid arthritis (P =.004, Mann-Whitney U test) No significant differences were observed in white blood cell counts, hemoglobin levels, platelet counts, erythrocyte sedimentation rate, or uric acid or aspartate aminotransferase levels at initial evaluation. CONCLUSIONS: Serum lactate dehydrogenase values may distinguish patients with malignant neoplasms from those with rheumatic disease early in the course of illness when symptoms and other laboratory values are not helpful.

Adolescent↗