NAD Oxidoreductase Research Articles

The Number of Genes for Lactate Dehydrogenase in Salmonid Fishes

Rainbow trout (Salmo gairdneri) that show polymorphism at the ldhHα locus for liver lactate dehydrogenase (LDH) (L-lactate: NAD oxidoreductase, EC 1.1.1.27) have been examined electrophoretically and immunologically. These results show that the ldhHα locus is active in two possible allelic forms, ldhHαA and ldhHαB, in many tissues such as heart, liver, kidney, eye, red blood cell, brain, and pyloric caeca. Quantitative microcomplement fixation tests support the contention that HαA and HαB subunits are allelic variants. These results, combined with electrophoretic studies on brook trout (Salvelinus fontinalis) polymorphic at the ldhHβ locus, lead us to conclude that all isozymes for LDH in salmonid fishes can be accounted for by the activity of four, and possibly five structural loci: ldhHα, ldhHβ, ldhC, ldhMα, and possibly ldhMβ. These results are in contrast to previous suggestions that the multiple lactate dehydrogenases of trout are encoded by 8 or 10 structural genes per haploid genome.

The Diurnal Rhythm of Enzymes in Human Red Cells

The activities of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, G6PD), 6-phosphogluconate dehydrogenase (6-phospho-d-gluconate: NADP oxidoreductase, 6PGD), hexokinase (ATP:D-hexose 6-phosphotransferase, HK), lactic dehydrogeanse (L-lactate: NAD oxidoreductase, LDH) and aspirate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, Asp.T) were determined in red blood cells of 11 healthy individuals. The determinations were carried out on samples drawn every 4 h over a 24 h period. The activities of G6PD, 6PGD, LDH and Asp.T exhibited a semi-circadian rhythm, namely, two peaks of activity during 24 h while HK activity demonstrated a true circadian rhythm. In addition a polymorphism of the G6PD and LDH activity patterns was observed. The implications of a biological clock in enucleated cells are discussed.

Subunit composition of alcohol dehydrogenase from Thea sinensis seeds and its substrate specificity for monoterpenes

Alcohol dehydrogenase (alcohol: NAD oxidoreductase, E.C. 1.1.1.1.) was purified from Thea sinensis seeds. Its M.W was 95000 and it was composed of two homogeneous subunits with MWs of 47000. The dissociation into subunits was caused by o-phenanthroline. Substrate specificity for monoterpene alcohols and aldehydes is discussed.

Horse liver aldehyde dehydrogenase. Purification and characterization of two isozymes.

Two isozymes of horse liver aldehyde dehydrogenase (aldehyde, NAD oxidoreductase (EC 1.2.1.3)), F1 and F2, have been purified to homogeneity using salt fractionation followed by ion exchange and gel filtration chromatography. The specific activities of the two isozymes in a pH 9.0 system with propionaldehyde as substrate were approximately 0.35 and 1.0 mumol of NADH/min/mg of protein for the F1 and F2 isozymes, respectively. The multiporosity polyacrylamide gel electrophoresis molecular weights of the F1 and F2 isozymes were approximately 230,000 and 240,000 respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave subunit molecular weight estimates of 52,000 and 53,000 for the F1 and F2 isozymes, respectively. The amino acid compositions of the two isozymes were found to be similar; the ionizable amino acid contents being consistent with the electrophoretic and chromatographic behavior of the two isozymes. Both isozymes exhibited a broad aldehyde specificity, oxidizing a wide variety of aliphatic and aromatic aldehydes and utilized NAD as coenzyme, but at approximately 300-fold higher coenzyme concentration could use NADP. The F1 isozyme exhibited a very low Km for NAD (3 muM) and a higher Km for acetaldehyde (70 muM), while the F2 isozyme was found to have a higher Km for NAD (30 muM) and a low Km for acetaldehyde (0.2 muM). The two isozymes showed similar chloral hydrate and p-chloromercuribenzoate inhibition characteristics, but the F1 isozyme was found to be several orders of magnittude more sensitive to disulfiram, a physiological inhibitor of acetaldehyde oxidation. Based on its disulfiram inhibition characteristics, it has been suggested that the F1 isozyme may be the primary enzyme for oxidizing the acetyldehyde produced during ethanol oxidation in vivo.

Saccharopine dehydrogenase. Substrate inhibition studies

In the direction of reductive condensation of alpha-ketoglutarate and lysine, saccharopine dehydrogenase (N6-(glutar-2-yl)-L-lysine:NAD oxidoreductase (lysine-forming) is inhibited by high concentrations of alpha-ketoglutarate and lysine, but not by NADH. NAD+ and saccharopine show no substrate inhibition in the reverse direction. Substrate inhibition by alpha-ketoglutarate and lysine is linear uncompetitive versus NADH. However, when the inhibition is examined with alpha-ketoglutarate or lysine as the variable substrate, the double reciprocal plots show a family of curved lines concave up. The curvature is more pronounced with increasing concentrations of the inhibitory substrate, suggesting an interaction of variable substrate with the enzyme form carrying the inhibitory substrate. These inhibition patterns, the lack of interaction of structural analogs of lysine such as ornithine and norleucine with the E-NAD+ complex (Fujioka M., and Nakatani, Y. (1972) Eur. J. Biochem. 25, 301-307), the identity of values of inhibition constants of alpha-ketoglutarate and lysine obtained with either one as the substrate inhibitor, and the substrate inhibition data in the presence of a reaction product, NAD+, are consistent with the mechanism that substrate inhibition results from the formation of a dead-end E-NAD+-alpha-ketoglutarate complex followed by the addition of lysine to this abortive complex.

Partial amino-acid sequence of NAD-specific glutamate dehydrogenase of Neurospora crassa.

Parts of the primary structure of the NAD-specific glutamate dehydrogenase [L-glutamate:NAD oxidoreductase (deaminating), EC 1.4.1.2] from Neurospora crassa are presented. Segments of the sequence representing 886 unique amino-acid residues have been determined; the largest contains 267 residues. There are only short regions of possible homology between this enzyme and the glutamate dehydrogenases of bovine liver or the NADP-specific enzyme of Neurospora. The large size of the subunit (116,000 molecular weight) of the NAD-specific glutamate dehydrogenase is unusual when compared to other known dehydrogenases.

ALCOHOL:NAD OXIDOREDUCTASE IN BRAIN OF RATS FROM A COLONY FED DILUTE ETHANOL FOR MANY GENERATIONS1,2

Abstract—Alcohol:NAD oxidoreductase (EC 1.1.1.1) was studied in brain cortex, hypothalamus, cerebellum and midbrain of adult and immature rats, and in the whole encephalon of neonatal rats. The rats used in this study were (i) from a colony which has been given 12% (v/v) aqueous ethanol as the only fluid for 54 generations (‘E.F.’ rats); (ii) rats removed from this colony after the forty‐eighth generation and thereafter fed water instead of the alcohol solution (‘E.F./H2O’ rats); and (iii) normal rats. Enzyme activity in the 20,000 g supernatant of tissue homogenates was measured by the method of Raskin and Sokoloff. Activity was found to be highest in neonatal rat brain and to decrease as the age increased. Activity in the hypothalamus of adult E.F. rats was significantly higher than that found in the same region of adult E.F./H2O rats. Immature rat cerebellum alcohol:NAD oxidoreductase activity was higher both in ‘E.F.’ and ‘E.F./H2O’ suggesting a possible genetic change be involved in this CNS region. It may be concluded that, with the exception of neonatal rats, ethanol consumption induces an increase in rat CNS alcohol :NAD oxidoreductase activity.

Factors limiting mitochondrial respiration in media of high solute content

The state-3 rate of respiration of rat-liver mitochondria was depressed in media containing KCl, sucrose, or mannitol at concentrations in excess of 125 mM. At equivalent concentrations, glucose caused less inhibition than sucrose or mannitol, and no inhibition was observed with glycine. These observations establish that solute inhibition of respiration is not a consequence of the reduced chemical potential of water in the system. The accumulation of succinate by mitochondria was not reduced by high sucrose concentrations. Sonication only partially relieved inhibition by sucrose or mannitol, and not at all that by KCl, and the evidence indicates that solute inhibition is not primarily an inhibition of substrate entry into mitochondria. Sucrose in the assay media inhibited succinate dehydrogenase [succinate: PMS oxidoreductase (EC.1. 3. 91)] and malate dehydrogenase [l-malate: NAD oxidoreductase (EC.1.1.1.37)] activities, but these inhibitions were less than those of succinate-and malate-dependent oxygen uptake by mitochondria. Disruption of the mitochondrial membrane by detergent abolished the inhibition of respiration by sucrose, and the evidence indicates that solute inhibits the functional capacity of the membrane-associated respiratory system.

Slow axoplasmic transport of mitochondria (mao) and lactic dehydrogenase in mammalian nerve fibers

The axoplasmic transport of the enzyme monoamine oxidase (MAO) (monoamine:O 2 oxidoreductase, (deaminating) EC 1.4.3.4), a marker for mitochondria, and lactic dehydrogenase (LDH) ( l-lactate_NAD oxidoreductase, EC 1.1.1.27), a soluble component of axoplasm, was studied in cat sciatic nerve. For both these enzymes a linear accumulation was found in the nerve proximal to ligation over a period of at least 20 h. In double-ligation experiments no evidence of a depletion of enzymes within the nerve segment was found over this period of time as would be the case if some portion of the enzymes was carried fast axoplasmic transport. Both the soluble protein enzyme LDH and the mitochondria, shown by MAO, are thus considered to be moved down the nerve by slow axoplasmic transport. Some differences in the two materials were seen in the greater fall in the level of MAO compared to LDH within the double-ligated segment over the succeeding period from 20 to 48 h. These changes are considered with respect to the transport filament model as modified to take into account slow axoplasmic transport.

Voltammetric measurement of reduced nicotinamide-adenine nucleotides and application to amperometric measurement of enzyme reactions

The electrochemical oxidations of reduced nicotinamide-adenine dinucleotide and reduced nicotinamide-adenine dinucleotide phosphate on platinum and carbon electrodes are described. Well defined voltammetric anodic waves are observed on carbon electrodes, with a linear relationship between peak height and concentration for 0–0.5m M NADH and NADPH. Amperometric methods for NAD oxidoreductase analyses by direct electrochemical oxidation of the reduced nucleotide have been developed for lactic dehydrogenase and ethanol in serum.

Determination of the Isotope Effect of the Enzymatic Oxidation of (R)Carnitine by Displacement of the Equilibrium via Mass Action

An isotope effect of the dehydrogenation of (R) Carnitine [(R) 3-hydroxy-4-trimethylaminobutyric acid hydrochloride] catalyzed by (R) carnitine dehydrogenase [(R) carnitine: NAD oxidoreductase E.C. 1.1.1.108] from Pseudomonas aeruginosa was measured at different temperatures. It was found that k1H/k3H does not vary greatly with changes of temperature. The value of 3 for k1H/k3H measured at small initial conversions strongly indicated that the rate limiting step of the oxidation of (R) carnitine is the cleavage of the C-H bond at C3.

Stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase from Bacillus subtilis

The stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase [(S)alanine : NAD oxidoreductase (EC 1.4.1.1)] from B. subtilis was investigated. The label at C-2 of (S) [2,3- 3H]alanine was enzymatically transferred to NAD, and the [4- 3H]NADH produced isolated and the stereochemistry at C-4 investigated. It was found that the label was exclusively located at the (R) position which indicates that (S)alanine dehydrogenase is an A-type enzyme. This result was confirmed in an alternate way by reducing enzymatically [4- 3H]NAD with non labeled (S)alanine and (S)alanine dehydrogenase and investigating the stereochemistry of the [4- 3H]NADH produced. As expected, the label was now exclusively located at the (S)position. This proves that (S)alanine dehydrogenase isolated from B. subtilis should be classified as an A-enzyme with regard to the stereochemistry of the hydrogen transfer to NAD.

THE EFFECTS OF HYPERKETONEMIA ON GLUTAMATE AND GLUTAMINE METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The effect of increased exposure to ketone bodies in the developing rat brain suggest that intrauterine and postnatal hyperketonemia lead to an altered metabolism of glutamine and glutamate. It is postulated that this effect is related to the delayed development of glutaminase (l‐glutamine amido‐hydrolase EC 3.5.1.2) and glutamate dehydrogenase (l‐glutamate: NAD oxidoreductase EC 1.4.1.2).The specific activities of glutamate dehydrogenase (GDH), glutaminase and glutamine synthetase (l‐glutamate: ammonia ligase EC 6.3.1.2) in the brains of newborn rats increased during early development. A positive correlation was observed between the specific activity of glutaminase and the concentration of glutamate in the brain as well as between the concentrations of blood and brain glutamine and glutamate in both control and hyperketonemic pups. This indicates a different degree of permeability and metabolism for glutamine and glutamate in the brain during the neonatal period, as compared to adulthood.In hyperketonemic pups, glutamine and glutamate metabolism were found to differ from that in control animals. The concentrations of glutamate were higher, and glutamine lower, in both the blood and brain as compared to that in controls. The concentrations of α‐ketoglutarate were also lower in their brain. In the brains of hyperketonemic and control pups, the concentration of malate was the same. During the first 3 weeks of life the increase of spec. act. of GDH and glutaminase was found to be suppressed in the brains of hyperketonemic pups. However, the spec. act. of glutamine synthetase was similar to that of the control pups.

Chromosomal location of mutations affecting the electrophoretic mobility of malate dehydrogenase in Escherichia coli K-12.

The structural locus for a soluble malate dehydrogenase (l-malate:NAD oxidoreductase, EC 1.1.1.37), mdh, lies about 1.2 min from aspB on the Escherichia coli chromosome in the sequence argG, aspB, mdh.

De novo synthesis and developmental control of the multiple gene-controlled malate dehydrogenase isozymes in maize scutella

In maize, both soluble and mitochondrial malate dehydrogenases ( l-malate:NAD oxidoreductase, EC 1.1.1.37) exist in multiple molecular forms (isozymes). Biochemical studies and genetic analysis showed that the maize malate dehydrogenase isozymes are not interconvertable conformational forms of a single gene product. Instead, they are determined by multiple genes which may reside on different chromosomes. Therefore, developmental expression of the various malate dehydrogenase isozymes was studied. During early development of the sporophyte (dry kernel to 10 days of germination), the total malate dehydrogenase activity in scutella increases through the first 5 days, peaks about the 6th day and decreases gradually thereafter. Both soluble and mitochondrial malate dehydrogenase isozymes exhibit similar activity profiles; however, the total mitochondrial malate dehydrogenase activity is only 60% of that in the cytosol. Density labeling experiments and atempts to detect the possible existence of “inactive malate dehydrogenase precursors” were performed. The results show that both soluble and mitochondrial malate dehydrogenase are synthesized in the scutella during germination and early growth of young maize seedlings. Accumulation of the maize malate dehydrogenase isozymes is probably controlled by synthesis as well as degradation instead of activation and inactivation of enzyme moieties. Correlation between the developmental pattern of the scutellar malate dehydrogenase isozymes and the physiological conditions of the young maize seedlings is discussed.

Investigations concerning the mode of action of 3,4-dihydroxybutyl-1-phosphonate on Escherichia coli.

Experiments were performed to evaluate the ability of the enzymes of Escherichia coli involved in glycerol 3-phosphate metabolism to recognize phosphonic acid analogues of the natural substrate. Neither the catabolic membrane-bound glycerol-3-phosphate dehydrogenase nor the acyl coenzyme A: glycerol-3-phosphate acyltransferase can use 3,4-dihydroxybutyl-1-phosphnate or 2,3-dihydroxypropyl-1-phosphonate are inhibitors of the reduction of dihydroxyactone phosphate as substrates. The 4-carbon phosphonic acid analogue does not exhibit inhibitory activity for either of these enzymes. While the 3-carbon phosphonic acid analogue has no inhibitory effect upon the catabolic dehydrogenase, it does appear to have a slight but reproducible inhibitory effect on the acyltransferase. Glycerol 3-phosphate and 3,4-dihydroxybutyl-1-phosphonate by glycerol 3-phosphate:NAD (P) oxidoreductase. rac-2,3-Dihydroxypropyl-1-phosphonate does not appear to be recognized by this enzyme. The apparent K-i for snglycerol 3-phosphate is 19 muM and for D-3,4-dihydroxybutyl-1-phosphonate it is 42 muM. In addition the glycerol 3-phosphate:NAD(P) oxidoreductase catalyzes the reduction of 4-hydroxy-3-oxobutyl-1-phosphonate (apparent K-m of 182 muM), a phosphonic acid analogue of dihydroxyacetone phosphate. 3,4-Dihydroxybutyl-1-phosphonate is both a competitive inhibitor (apparent Ki of 740 muM) and a substrate (apparent K-m of 450 muM) for the CDP-diglyceride: glycerol 3 phosphate phosphatidyltransferase but it has no effect upon CDP-diglyceride:L-serine phosphatidyltransferase. The relationship ofthese in vitro studies to in vivo investigations is discussed.

Regulatory Properties of Purified 3-Phosphoglycerate Dehydrogenase from Bacillus subtilis

3-Phosphoglycerate dehydrogenase (3-phosphoglycerate:NAD oxidoreductase, EC. 1.1.1.95) was purified from Bacillus subtilis by conventional methods. The final preparation was homogeneous by electrophoretic analysis and had a sedimentation constant of 6.3 S. On the basis of gel filtration data the enzyme had a molecular weight of about 166000. The plot of velocity versus phosphoglycerate concentration was biphasic while similar plots for hydroxypyruvate phosphate and NADH were the conventional hyperbolic type. The enzyme was specifically inhibited by serine. The inhibition was time dependent, requiring several minutes incubation before a constant level of inhibition was achieved. Serine inhibition was of the "mixed type" with respect to 3-phosphoglycerate and Hill plots of these data had slopes that approached 2. Desensitization of the enzyme to serine inhibition was achieved by incubation in the absence of dithiothreitol. The desensitized enzyme was different from the native enzyme in fluoresence properties, sedimentation characteristics and in the absence of the biphasic phosphoglycerate saturation curve. Evidence was obtained for the participation of sulphydryl groups in the changes in protein structure responsible for serine inhibition as well as the dehydrogenase activity of the enzyme.

Blood Leucocyte Enzymes

The activities of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, G6PD), 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP oxidoreductase, 6PGD), hexokinase (ATP: D-hexose 6-phosphotransferase, Hx), lactate dehydrogenase (D-lactate: NAD oxidoreductase, LDH). glutamate oxaloacetate transaminase (L-aspartate: 2 oxoglutarate aminotransferase, GOT) and dihydrofolate reductase (DHFR) were measured at 8 a.m. in leucocytes of healthy individuals and patients with chronic myeloid leukaemia (CML), chronic lymphatic leukaemia (CLL), myelofibrosis with myeloid metaplasia and polycythaemia vera. In view of the heterogeneity of the leucocyte populations in these conditions, the enzyme activities were correlated to the number of immature cells in CML and to the percentage of lymphocytes in CLL. No differences in the enzyme activities were found between the white cells of healthy individuals, myelofibrosis with myeloid metaplasia and polycythaemia vera. In CML the activities of all enzymes except GOT correlated directly with the number of immature cells; an inverse correlation with the number of lymphocytes was observed in CLL. GOT was the only enzyme whose activity correlated with the number of lymphocytes in the cell suspension. Furthermore, a significantly higher activity of this enzyme was found in Ficoll-isolated CLL lymphocytes as compared to normal lymphocytes.

Initial velocity and product inhibition studies on L-iditol:NAD oxidoreductase.

Initial velocity and product inhibition studies on L-iditol:NAD oxidoreductase.

Effect of testosterone-estradiol-binding globulin in the enzymic oxidoreduction of 17-oxygenated c19 steroids.

The effect of both testosterone-estradiol-binding globulin (TeBG) and albumin on enzymic oxidoreduction of four 17-oxygenated C19 steroids by bacterial 17beta-hydroxysteroid:NAD oxidoreductase from Pseudomonas testosteroni was investigated. The decreased yields of products under presence of TeBG were found in both directions of reversible enzymic reaction. This finding was unexpected in the case of enzymic reduction in which the opposite effect could be assumed with respect of high affinity of the product but not the substrate to TeBG. Kinetically, the competition between enzyme and binding protein for the substrate occurs at enzymic oxidation, whereas the mechanism resembling non-competitive inhibition operates in the enzymic reduction.