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)

Large numbers of taste buds are distributed over the body surface of the channel catfish ictalurus punctatus, with the barbels having an especially high density. L-Alanine, as well as certain other amino acids, are taste stimuli in this animal. Epithelial tissue obtained by gentle scraping of the barbel surface was fractionated by differential centrifugation. A sedimentable fraction (P2) was prepared that was enriched in L[OH]alanine binding activity, the plasma membrane marker enzyme 5'-nucleotidase, and the mitochondrial marker succinate cytochrome c reductase, but not the microsomal marker NADH cytochrome c redu.ctase. Binding of L-[OH]alanine was measured using a Millipore filter method in which correction for non-specific binding was also determined. Time, temperature, and pH for measuring binding activity were established. At the optimal pH of 7.8, the KD for L-alanine is 4.8 X 10(-6) M. The first order dissociation rate constant at 6 degrees is 3.8 X 10(-4) s-1 and at 24 degrees it is 12.1 X 10(-4) s-1. The second order rate constant for association is between 10(2) and 10(3) M-1 S-1. Reversibility of the binding interaction was also demonstrates by the rapid displacement of bound L-[3H]alanine by a large excess of unlabeled L-alanine. That the binding does not represent incorporation into protein was confirmed by the lack of effect of puromycin. The amounts bound of several other chemostimulatory amino acids werealso determined.
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PMID:Biochemical studies of tast sensation. Binding of L-[3H]alanine to a sedimentable fraction from catfish barbel epithelium. 0 Apr 3

L-alanine dehydrogenase, (L-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.1) synthesis in a thermophilic bacillus was found to be subjected to regulatory control. Addition of L- and D-alanine and L-serine to cultures growing in the presence of either succinate or pyruvate, induced an accelerated synthesis of the alanine dehydrogenase enzyme. Synthesis of the enzyme was dependent on the presence of inducer during growth and was arrested by addition of glucose. Catabolite repression by glucose was abolished by limiting the ammonium concentration during growth. The apparent Km values of the substrates involved in alanine dehydrogenase activity are as follows (M): NH4+, 4-10(-2); pyruvate, 5-10(-4); NADH, 6-10(-5); L-alanine, 3.1-10(-3) and NAD, 2-10(-4). Alanine dehydrogenase activity was measurable at temperatures below the minimal growth temperature (at 25 degrees C) and the highest activity was found at 65 degrees C; heat denaturation occurred at 80 degrees C.
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PMID:Regulatory control and function of alanine dehydrogenase from a thermophilic bacillus. 0 88

Cytoplasmic membranes of Bacillus subtilis, grown in complex medium containing glucose, were fractionated into three membrane subfractions [light band (1.155 - 1.158 g/cm3); medium band (1.181 - 1.183 g/cm3); heavy band (1.21 - 1.25 g/cm3)] by sucrose density gradient centrifugation. Among these subfractions, the light and medium bands consisted mainly of membranes but the heavy band consisted of an irregular arrangement or aggregate of small globular protein components of 5 - 8 nm in diameter. We named this H-protein. H-protein formed trilamellar unit membrane structure when combined with lipid. In pulse-labeling and pulse-chase experiments with radioactive leucine, it was found that H-protein consisted of the newest membrane protein synthesized in the cells and the label incorporated into H-protein was shifted into light and medium band of the membranes during the chase. Cytochromes were not found in H-protein. However, when H-protein was incubated with haem alpha and protohaem, these compounds were incorporated into the apoproteins of the cytochromes present in H-protein and form cytochromes a and b. Cytochromes were also formed in H-protein which were isolated from the cells grown in the presence of haemin (haemin-grown H protein). Succinate dehydrogenase activity was increased about 4-fold by combining H-protein or haemin-grown H protein with lipid. H-protein had no cytochrome oxidase activity; however, haemin-grown H protein was found to have some of the activity and this was increased about 4-fold by combining the protein with lipid. Haemin-grown H protein was also found to form succinate: cytochrome c oxidoreductase when combined with lipid and vitamin K2. On the other hand, succinate oxidase was required for the addition of lipid, vitamin K2 and cytochrome c. NADH oxidase was also found in haemin-grown H protein and was activated about 9-fold in constituted reaction systems. Vesicles formed by haemin-grown H protein and lipid, could accumulate alanine and proline by addition of NADH or reduced phenazine methosulfate. Alanine and proline was also accumulated into the vesicles when transport energy was supplied as a membrane potential introduced by K+-diffusion via valinomycin. These results would indicate that H-protein contains the apoprotein of cytochromes, and a carrier involved in the active transport of alanine and proline.
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PMID:Isolation and characterization of hydrophobic proteins (H proteins) in the membrane fraction of Bacillus subtilis. Involvement in membrane biosynthesis and the formation of biochemically active membrane vesicles by combining H proteins with lipid. 18 52

Ratios of yields of cholesterol in different media with different metabolites (200 estimations) show that if the initial normal quantity in a system is X, glucose makes it 4X which is then reduced by the amino acids and ascorbic acid. Alanine, Serine, Threonine, Cysteine, Cystine and Lysine reduce it to 2X. Glycine, Valine, Leucine, Aspartic, Phenylalanine, Tyrosine, Tryptophan roughly increase it to 6X. Other amino acids are intermediate between the above sets. Balanced amino acids and fatty acids keep up the glucose value 4X but the individual amino acids behave as above. Ascorbic acid reduces the quantity of cholesterol everywhere from one third to one half bringing it back to normal X. Since it is not directly involved in the cholesterol synthesis, it must be acting through the pyridine nucleotides. The interesting feature is that the C/H ratio in the amino acids corresponds to the yield of cholesterol with respect to glucose saline yield = 1. Mechanism of metabolic shifts has been discussed on the empirical basis as also on the basis of the change in ratios of NAD+ (NADP+) to NADH2 (NADPH2) through ascorbic acid.
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PMID:Studies on the cholesterol synthesis in the human adipose tissue. II. Mechanism of metabolic shifts and regulation of cholesterol through ascorbic acid. 22 11

1. L-Alanine dehydrogenase (L-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.1) was purified about 500-fold from Halobacterium salinarium. 2. The enzyme appears to be homogeneous in polyacrylamide gel electrophoresis. The apparent molecular weight is about 60 000. 3. Activity and stability of the enzyme are largely affected by different salts. Full activity of the NADH-dependent reductive amination of pyruvate occurs at 4.3 M NaCl. This activation can be achieved also by KCl and several other salts instead of NaCl. 4. The NAD+-dependent oxidative deamination of L-alanine occurs only in the presence of high concentrations of KCl. This reaction is not stimulated by NaCl. The Km values for the substrates NADH, pyruvate and NH+4 are also salt dependent. 5. The thermal stability of the enzyme is considerably higher in the presence of high concentrations of NaCl than in the presence of KCl. 6. The enzyme is completely inactivated by the removal of salt. Full reactivation is achieved by addition of salt in the presence of 2-mercaptoethanol. Inactivation proceeds about ten times faster than reactivation. The inactivation after the withdrawal of salt and the reactivation following the readdition of salt show a characteristic hysteresis loop.
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PMID:Purification and properties of alanine dehydrogenase from Halobacterium salinarium. 48 98

1. The bacterial distribution of alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating, EC 1.4.1.1) was investigated, and high activity was found in Bacillus species. The enzyme has been purified to homogeneity and crystallized from B. sphaericus (IFO 3525), in which the highest activity occurs. 2. The enzyme has a molecular weight of about 230 000, and is composed of six identical subunits (Mr 38 000). 3. The enzyme acts almost specifically on L-alanine, but shows low amino-acceptor specificity; pyruvate and 2-oxobutyrate are the most preferable substrates, and 2-oxovalerate is also animated. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. 4. The enzyme is stable over a wide pH range (pH 6.0--10.0), and shows maximum reactivity at approximately pH 10.5 and 9.0 for the deamination and amination reactions, respectively. 5. Alanine dehydrogenase is inhibited significantly by HgCl2, p-chloromercuribenzoate and other metals, but none of purine and pyrimidine bases, nucleosides, nucleotides, flavine compounds and pyridoxal 5'-phosphate influence the activity. 6. The reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by ammonia and pyruvate, and the products are released in the order of L-ALANINE AND NAD+. The Michaelis constants are as follows: NADH (10 microM), ammonia (28.2 mM), pyruvate (1.7 mM), L-alanine (18.9 mM) and NAD+ (0.23 mM). 7. The pro-R hydrogen at C-4 of the reduced nicotinamide ring of NADH is exclusively transferred to pyruvate; the enzyme is A-stereospecific.
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PMID:Purification and properties of alanine dehydrogenase from Bacillus sphaericus. 48 97

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.
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PMID:Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism. 239 Sep 93

L-Alanine dehydrogenase was partially purified from the mycelial extracts of Cunninghamella elegans. The purified enzyme was fractionated by TEAE-cellulose column chromatography into two fractions (subunits) designated as fractions I and II. The activity of both fractions in the aminating reaction is 8 times higher than the activity of the deaminating reaction. pH-Optimal for reductive amination of pyruvate by both fractions were 8 and 10 for oxidative deamination of L-alanine. The Km values of fractions I and II for L-alanine, NAD+, pyruvate, NH4+ and NADH were estimated. Both fractions of L-alanine dehydrogenase were absolutely specific for L-alanine in the oxidative deamination reaction. Maximal activity of both fractions occurred at 40 degrees C. Inhibition by iodoacetate and activation by SH-compounds suggest that sulfhydryl groups may participate in enzyme activity. The 2 fractions were activated with Co2+, Fe2+, Ca2+, Mg2+ and Mn2+ ions, while Zn2+ inhibited both fractions. Stability of the enzyme under different conditions was investigated.
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PMID:Partial purification and properties of Cunninghamella elegans L-alanine dehydrogenase. 280 Jul 49

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.
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PMID:Alanine dehydrogenase from Streptomyces fradiae. Purification and properties. 291 62

Alanine dehydrogenase from Bacillus cereus, a non-allosteric enzyme composed of six identical subunits, was purified to homogeneity by chromatography on blue-Sepharose and Sepharose 6B-CL. Like other pyridine-linked dehydrogenases, alanine dehydrogenase is inhibited by Cibacron blue, competitively with respect to NADH and noncompetitively with respect to pyruvate. The enzyme was inactivated by 0.1 M glycine/HCl (pH 2) and reactivated by 0.1 M phosphate (pH 8) supplemented with NAD+ or NADH. The reactivation was characterized by sigmoidal kinetics indicating a complex mechanism involving rate-limiting folding and association steps. Cibacron blue interfered with renaturation, presumably by competition with NADH. Chromatography on Sepharose 6B-CL of the partially renatured alanine dehydrogenase led to the separation of several intermediates, but only the hexamer was characterized by enzymatic activity. By immobilization on Sepharose 4B, alanine dehydrogenase from B. cereus retained 66% of the specific activity of the soluble enzyme. After denaturation of immobilized alanine dehydrogenase with 7 M urea, 37% of the initial protein was still bound to Sepharose, indicating that on the average the hexamer was attached to the matrix via, at most, two subunits. The ability of the denatured, immobilized subunits to pick up subunits from solution shows their capacity to fold back to the native conformation after urea treatment. The formation of "hybrids" between subunits of enzyme from B. cereus and Bacillus subtilis demonstrates the close resemblance of the tertiary and quaternary structures of alanine dehydrogenases from these species.
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PMID:Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus. 310 22


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