Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT02288 (NADH)
 21,914 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this method, blood is collected in ammonium heparinized microhematocrit tubes and lactate is directly determined in the plasma, separated within 15 min from the erythrocytes. Lactate is assayed by mixing 10 mul of sample with NAD+ and lactate dehydrogenase in tris(hydroxymethyl)aminomethane hydrazine buffer. The rate of increase in absorbance of the NADH formed, measured at 340 nm, is proportional to lactate concentration. The assay is complete in 4 min and absorbance is linearly related to concentration from 0.625 to 15 mmol/liter. Analytical recoveries of lactate added to plasma averaged 104% (range, 91-116%). Results compared well for plasma samples analyzed by this method with the CentrifiChem and the Du Pont aca.
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PMID:Rapid kinetic measurement of lactate in plasma with a centrifugal analyzer. 0 Jan 61

Re-investigating the accuracy of the commonly used values for molar absorptivities (epsilon) of beta-NADH and beta-NADPH at Hg 334, Hg 365, or 340 nm, we obtained the following results: The maximum of absorbance of NADH is shifted from about 340 nm at 0 degrees C to about 338.5 nm at 38 degrees C; the corresponding maxima of NADPH are located at about 0.5-nm longer wavelengths. In addition, the absorption curves of both coenzymes broaden with increasing temperature. For these reasons, the epsilon-values of NADH and NADPH are generally different from each other, and are temperature-dependent. Only at 334 nm are they almost identical and nearly independent of temperature. Therefore this wavelength is recommended for precise measurements. The epsilon-values of these coenzymes are influenced by ionic strength and pH. To determine the absolute values of the molar absorptivities, we performed the glutamate dehydrogenase or lactate dehydrogenase assay with carefully purified 2-oxoglutaric acid or pyruvic acid in the presence of excess coenzyme. The purity of the substrates was checked through differential scanning calorimetry, moisture analysis, gas-liquid chromatography, gas chromatography in combination with mass spectrometry, and nuclear magnetic resonance spectroscopy. The epsilon-values observed under the various conditions are about 1-7% higher than those currently used.
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PMID:Molar absorptivities of beta-NADH and beta-NADPH. 0 89

1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2. In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results.
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PMID:Pig heart lactate dehydrogenase. Binding of pyruvate and the interconversion of pyruvate-containing ternary complexes. 0 75

The kinetics of pyruvate transport across the isolated red blood cell membrane were studied by a simple and precise spectrophotometric method: following the oxidation of NADH via lactate dehydrogenase trapped within resealed ghosts. The initial rate of pyruvate entry was linear. Influx was limited by saturation at high pyruvate concentration. Pyruvate influx was greatly stimulated by increasing ionic strength in the outer but not the inner aqueous compartment. The Km ranged from 15.0 mM at mu = 0.05 to 3.7 mM at mu = 0.01, while the V went from 0.611 - 10(15) to 0.137 - 10(-15) mol - min-1 - ghost-1. Ionic strength was shown to affect the translocation step and not pyruvate binding. The energy of activation of pyruvate flux into resealed ghosts was 25 kcal/mol, similar to that found in intact red blood cells. Inhibitors of pyruvate influx included such anions as thiocyanate, chloride, bicarbonate, alpha-cyanocinnamate, salicylate and ketomalonate (but not acetate); noncompetitive inhibitors were phloretin, 1-fluoro-2,4-dinitrobenzene, 4-acetamido-4'-isothiocyanate-stilbene-2,2'-disulfonic acid and o-phenanthroline/CuSO4 mixtures. The last reagent, known to induce disulfide links in certain membrane proteins, blocked the ionic strength stimulation of pyruvate influx in this study.
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PMID:Pyruvate flux into resealed ghosts from human erythrocytes. 0 47

The chain oxidation of lactate dehydrogenase-bound NADH initiated by superoxide radicals and propagated by oxygen was studied with pulse radiolysis. The kinetic parameters were re-evaluated in a system with carefully purified reagents (water and other chemicals) and in the presence of EDTA. The rate constant for the oxidation of the enzyme-bound NADH by O2- is calculated from the observed pseudo-first order disappearance of NADH and the chain length (molecules of NADH oxidized per O2- anion generated in the pulse). It is (1.0 +/- 0.2) X 10(5) M-1 S-1, consistent within a 13-fold variation in lactate dehydrogenase. NADH complex concentration and with varying chain length up to 6.1. Based on experiments with varying pH values from 4.5 to 9.0, the rate constant for oxidation of enzyme-bound NADH by HO2 is estimated to be 2.0 X 10(6) M-1 S-1.
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PMID:Re-evaluation of the kinetics of lactate dehydrogenase-catalyzed chain oxidation of nicotinamide adenine dinucleotide by superoxide radicals in the presence of ethylenediaminetetraacetate. 0 64

Analyzes were made on muscle samples taken from the lateral part of the m. quadriceps femoris of man (lactate, pyruvate, and pH) on venous blood (lactate, pyruvate) and on capillary blood (pH). Samples were taken at rest, immediately after termination of dynamic exercise and during 20 min recovery from exhaustive dynamic exercise. Muscle pH decreases from 7.08 atrest to 6.60 at exhaustion. Decrease in muscle pH was linearly related to muscle content of lactate + pyruvate. The relationship was slightly different from what has been obtained after isometric exercise and this difference was ascribed to acid-base exchange with the blood during dynamic exercise. Lactate content was highly elevated in muscle after exercise and the concentration was 2-3 times higher than in blood. Pyruvate content was, however, only slightly higher than that at rest. During recovery, lactate content of muscle decreased exponentially with respect to time, whereas pyruvate content increased. The half-time of lactate decrease was 9.5 min. From the lactate dehydrogenase equilibrium relative values on NADH/NAD ratio could be calculated. It was found that NADH/NAD was highly increased after exercise and that it had not returned to the basal value after 20 min recovery.
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PMID:Lactate content and pH in muscle obtained after dynamic exercise. 1 43

Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is suggested that, under anaerobic conditions, muscles of marine invertebrates form lactate and/or octopine or succinate (or similar end product) according to the activities of the enzymes present in the muscles (see above). The muscles investigated possess low activities of cytosolic glycerol 3-phosphate dehydrogenase, which indicates that glycerol phosphate formation is quantitatively unimportant under anaerobic conditions, and low activities of mitochondrial glycerol phosphate dehydrogenase, which indicates that the glycerol phosphate cycle is unimportant in the re-oxidation of glycolytically produced NADH in these muscles under aerobic conditions. Conversely, high activities of glutamate-oxaloacetate transaminase are present in some muscles, which indicates that the malate-aspartate cycle may be important in oxidation of glycolytically produced NADH under aerobic conditions. 3. High activities of nucleoside diphosphate kinase were found in muscles that function for prolonged periods under anaerobic conditions (e.g...
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PMID:The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates. 1 83

Chicken liver lactate dehydrogenase (L-lactate : NAD+ oxidoreductase, EC 1.1.1.27) irreversibly catalyses the oxidation of glyoxylate (hydrated form) (I) to oxalate (pH = 9.6) and the reduction of (non-hydrated form) (II) to glycolate (pH = 7.4). (I) attaches to the enzyme in the pyruvate binding site and (II) attaches to the enzyme at the L-lactate binding site. The oxidation of (I) (pH = 9.6) is adapted to the following mechanism: (see book). The abortive complexes, E-NADH-I and E-NAD+-II, are responsible for the inhibition by excess substrate in the reduction and oxidation systems, respectively. When lactate dehydrogenase and NAD+ are preincubated, E-NAD+- NAD+ appears and causes inhibition by excess NAD+ in the glyoxylate-lactate dehydrogenase-NAD+ and L-lactate-lactate dehydrogenase-NAD+ systems; the second NAD+ molecule attaches to the enzyme at the L-lactate binding site.
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PMID:Kinetic formulations for the oxidation and the reduction of glyoxylate by lactate dehydrogenase. 1 38

Methyl methanethiosulphonate was used to produce a modification of the essential thiol group in lactate dehydrogenase which leaves the enzyme catalytically active. Methyl methanethiosulphonate produced a progressive inhibition of enzyme activity, with 2mM-pyruvate and 0.14mM-NADH as substrates, which ceased once the enzyme had lost 70-90% of its activity. In contrast, with 10mM-lactate and 0.4mM-NAD+ as substrates the enzyme was virtually completely inhibited. The observed inhibition was critically dependent on the chosen substrate concentration, since methanethiolation with methyl methanethiosulphonate resulted in a large decrease in affinity for pyruvate. At 0.14mM-NADH, methanethiolation increased the apparent KmPyr from from 40micronM for the control enzyme to 12mM for the modified enzyme. Steady-state kinetics showed that there was not a statistically significant change in either KmNADH or KsNADH. At saturating NADH and pyruvate concentrations, the Vmax. was virtually unaffected for the methanethiolated enzyme. However, a decrease in Vmax. was observed when the modified enzyme was incubated in dilute solution. The modification of lactate dehydrogenase by methyl methanethiosulphonate involved the active site, since inhibition was completely prevented by substrate-analogue pairs such as NADH and oxamate or NAD+ and oxalate. The formation of complexes between methanethiolated lactate dehydrogenase and substrates or substrate analogues can also be shown by re-activation experiments. The methanethiolated enzyme was re-activated in a time-dependent reaction by dithiothreitol and this was prevented by oxamate, by NADH and by NADH plus oxamate in increasing order of effectiveness. The results of this work are interpreted in terms of a role for the essential thiol group in the binding of substrates.
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PMID:Modification of pig heart lactate dehydrogenase with methyl methanethiosulphonate to produce an enzyme with altered catalytic activity. 1 52

The L-(+)-lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) of Streptococcus lactis C10, like that of other streptococci, was activated by fructose 1,6-diphosphate (FDP). The enzyme showed some activity in the absence of FDP, with a pH optimum of 8.2; FDP decreased the Km for both pyruvate and reduced nicotinamide adenine dinucleotide (NADH) and shifted the pH optimum to 6.9. Enzyme activity showed a hyperbolic response to both NADH and pyruvate in all the buffers tried except phosphate buffer, in which the response to increasing NADH was sigmoidal. The FDP concentration required for half-maximal velocity (FDP0.5V) was markedly influenced by the nature of the assay buffer used. Thus the FDP0.5V was 0.002 mM in 90 mM triethanolamine buffer, 0.2 mM in 90 mM tris(hydroxymethyl)aminomethanemaleate buffer, and 4.4 mM in 90 mM phosphate buffer. Phosphate inhibition of FDP binding is not a general property of streptococcal lactate dehydrogenase, since the FDP0.5V value for S. faecalis 8043 lactate dehydrogenase was not increased by phosphate. The S. faecalis and S. lactis lactate dehydrogenases also differed in that Mn2+ enhanced FDP binding in S. faecalis but had no effect on the S. lactis dehydrogenase. The FDP concentration (12 to 15 mM) found in S. lactis cells during logarithmic growth on a high-carbohydrate (3% lactose) medium would be adequate to give almost complete activation of the lactate dehydrogenase even if the high FDP0.5V value found in 90 mM phosphate were similar to the FDP requirement in vivo.
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PMID:Fructose 1,6-diphosphate-activated L-lactate dehydrogenase from Streptococcus lactis: kinetic properties and factors affecting activation. 1 95


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