Role Of NAD Research Articles

Cleavage of spermidine as the first step in deoxyhypusine synthesis. The role of NAD.

The biosynthesis of deoxyhypusine (N-(4-aminobutyl)lysine) occurs by the transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in a precursor of eukaryotic translation initiation factor 4D (eIF-4D). Deoxyhypusine synthase, the enzyme that catalyzes this reaction, was purified approximately 700-fold from rat testis. The Km values for the substrates, spermidine, the eIF-4-D precursor protein, and NAD+, were estimated as approximately 1, 0.08, and 30 microM, respectively. After incubation of partially purified enzyme with [1,8-3H]spermidine, NAD+, and the eIF-4D precursor, equal amounts of radioactivity were found in free 1,3-diaminopropane and in protein-bound deoxyhypusine. However, when the protein substrate (eIF-4D precursor) was omitted, radioactivity was found in 1,3-diaminopropane and in delta 1-pyrroline in nearly equal quantities, providing evidence that the cleavage of spermidine occurs, albeit at a slower rate, in the absence of the eIF-4D precursor. That NAD+, which is required for this reaction, functions as the hydrogen acceptor was demonstrated by the fact that radioactivity from spermidine labeled with 3H at position 5 is found in NADH as well as in delta 1-pyrroline. Transfer of this hydrogen from spermidine to the re face of the nicotinamide ring of NAD+, as determined by the use of dehydrogenases of known stereospecificity, defines the first step of deoxyhypusine synthesis as a pro-R, or A, stereospecific dehydrogenation. Based on these findings, an enzyme mechanism involving imine intermediate formation is proposed.

Purification, properties, and metabolic roles of NAD+-glutamate dehydrogenase in Clostridium botulinum 113B.

Cell-free extracts of proteolytic strains of Clostridium botulinum types A, B and F (group I) were found to have unusually high specific activities of NAD+-dependent L-glutamate dehydrogenase (NAD-GDH). In comparison, nonproteolytic strains of types B, E and F (group II) had low specific activities. The enzyme was purified 131-fold from C. botulinum 113B to a final specific activity of greater than 1,092 mumol x min-1 x mg protein-1. The enzyme is a hexamer of a polypeptide of Mr = 42,500, and the native molecular weight is 250,800. The apparent Km values for substrates were 5.3 mM for glutamate and 0.028 mM for NAD+ in the deamination reaction, and 7.2 mM for alpha-ketoglutarate, 243 mM for NH4+ and 0.028 mM for NADH in the reverse reaction. NADP+ did not serve as a hydrogen acceptor for the enzyme. Activity in the animation direction was inhibited by fumarate, oxalacetate, aspartate, glutamate and glutamine. The results suggest that GDH is important in group I (proteolytic) C. botulinum to generate alpha-ketoglutarate as a substrate for transamination reactions. We have also found that the high activity decreases significantly when cells are exposed to sodium chloride. Therefore GDH probably has several important physiological roles in group I proteolytic C. botulinum.

Role of NAD+-linked malic enzymes as regulators of the pool size of tricarboxylic acid-cycle intermediates in the perfused rat heart

Role of NAD+-linked malic enzymes as regulators of the pool size of tricarboxylic acid-cycle intermediates in the perfused rat heart

Glycerol and dihydroxyacetone dissimilation in Desulfovibrio strains

During growth on glycerol two marine Desulfovibrio strains that can grow on an unusually broad range of substrates contained high activities of glycerol kinase, NAD(P)-independent glycerol 3-phosphate dehydrogenase and the other enzymes necessary for the conversion of dihydroxyacetone phosphate to pyruvate. Glycerol dehydrogenase and a specific dihydroxyacetone kinase were absent. During growth on dihydroxyacetone, glycerol kinase is involved in the initial conversion of this compound to dihydroxyacetone phosphate which is then further metabolized. Some kinetic properties of the partially purified glycerol kinase were determined. The role of NAD as electron carrier in the energy metabolism during growth of these strains on glycerol and dihydroxyacetone is discussed. Glycerol also supported growth of three out of four ‘classical’ Desulfovibrio strains tested. D. vulgaris strain Hildenborough grew slowly on glycerol and contained glycerol kinase, glycerol 3-phosphate dehydrogenase and enzymes for the dissimilation of dihydroxyacetone phosphate. In D. gigas which did not grow on glycerol the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase were absent in lactate-grown cells.

Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases

The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH: cytochrome b 5 (EC 1.6.2.2) and NADPH: cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD + and NADP +, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O 2 − than O 2 2−. We suggest that a ferroheme-O 2 and various heme-oxy radical complexes (mainly ferroheme-O 2 − complex) play a crucial role in NAD(P)H oxidation.

Nicotinamide adenine dinucleotide and renal response to parathyroid hormone.

We tested the hypothesis that NAD plays a role in the cellular mechanism of action of parathyroid hormone (PTH) on phosphate transport. PTH significantly increased urinary cyclic AMP and phosphate excretions in thyroparathyroidectomized rats. The NAD+/NADH ratio, but not total NAD content, in renal cortical tissue was significantly higher in rats infused with PTH than in controls. The results demonstrate that the phosphaturic effect of PTH is associated with a shift in the renal cortical NADH to NAD+ and provides evidence for a role for NAD+ in the cellular regulation of phosphate transport.

Possible involvement of the enhanced tryptophan pyrrolase activity in the corticosterone- and starvation-induced increases in concentrations of nicotinamide-adenine dinucleotides (phosphates) in rat liver.

1. Deoxycorticosterone, which does not enhance tryptophan pyrrolase activity, also fails to alter the concentrations of the NAD(P) couples in livers of fed rats, whereas corticosterone increases both pyrrolase activity and dinucleotide concentrations. 2. Starvation of rats increases serum corticosterone concentration, lipolysis, tryptophan availability to the liver, tryptophan pyrrolase activity and liver [NADP(H)]. Glucose prevents all these changes. 3. The beta-adrenoceptor-blocking agent propranolol prevents the starvation-induced lipolysis and the consequent increase in tryptophan availability to the liver, but does not influence the increase in serum corticosterone concentration, liver pyrrolase activity and [NADP(H)]. 4. Actinomycin D, which prevents the starvation-induced increases in liver pyrrolase activity and [NADP(H)], does not affect those in serum corticosterone concentration and tryptophan availability to the liver. 5. Allopurinol, which blocks the starvation-induced enhancement of pyrrolase activity, also abolishes the increases in liver [NADP(H)], but not those in serum corticosterone concentration or tryptophan availability to the liver. 6. It is suggested that liver tryptophan pyrrolase activity plays an important role in NAD+ synthesis from tryptophan in the rat.

Biochemical characteristics of non‐streptomycin‐producing mutants of Streptomyces griseus I. Role of NAD (P)‐glycohydrolase in cell differentiation

AbstractFive non‐streptomycin‐producing mutants of an industrial strain of Streptomyces griseus lacking aerial mycelium formation were compared with their genetic ancestor and another producing mutant with regard to the NAD(P)‐glycohydrolase activity during cultivation on different media. By contrast to producing strains, all the Str− Amy− mutants displayed much lower mycelial and extracellular levels of enzyme, thus confirming earlier contentions concerning its involvement in the regulation of aerial mycelium formation. A tentative model is shown of the mechanism of control by phospho‐adenosinediphospho‐ribose of the intermediary metabolism at the sites of the citric acid cycle and the catabolism of the carbohydrates during distinct stages of cell differentiation. On the other hand, in the producing strain the biosynthesis of NAD(P)‐glycohydrolase was demonstrated to depend on the regime of fermentation. Repeated stages of submerged cultivation in stirred fermentors resulted in suppression of enzyme formation without concomitant change of the mycelial capacity to produce streptomycin. This suggests that there is no direct involvement of NAD(P)‐glycohydrolase in the control of antibiotic biosynthesis.

Biochemical characteristics of non-streptomycin-producing mutants of Streptomyces griseus. I. Role of NAD (P)-glycohydrolase in cell differentiation.

Five non-streptomycin-producing mutants of an industrial strain of Streptomyces griseus lacking aerial mycelium formation were compared with their genetic ancestor and another producing mutant with regard to the NAD(P)-glycohydrolase activity during cultivation on different media. By contrast to producing strains, all the Str- Amy- mutants displayed much lower mycelial and extracellular levels of enzyme, thus confirming earlier contentions concerning its involvement in control by phospho-adenosinediphospho-ribose of the intermediary metabolism at the sites of the citric acid cycle and the catabolism of the carbohydrates during distinct stages of cell differentiation. On the other hand, in the producing strain the biosynthesis of NAD(P)-glycohydrolase was demonstrated to depend on the regime of fermentation. Repeated stages of submerged cultivation in stirred fermentors resulted in suppression of enzyme formation without concomitant change of the mycelial capacity to produce streptomycin. This suggests that there is no direct involvement of NAD(P)-glycohydrolase in the control of antibiotic biosynthesis.

Role of NAD + in the stimulation of protein synthesis in rabbit reticulocyte lysates

The stimulation of protein synthesis by NAD + in rabbit reticulocyte lysates has been reported. [Lennon M. B., Wu, J., and Suhadolnik, R. J., (1976) Biochem. Biophys. Res. Commun. 72, 530–538]. NAD + can replace the creatine phosphate-creatine phosphokinase ( CP CPK ) energy regenerating system normally used in in vitro protein synthesizing systems. The replacement of CP CPK by NAD + is optimal at 37 °C. A significant lag in the rate of protein synthesis with NAD + is observed with decreasing temperatures. Analysis of the adenylate energy charge with NAD + shows an initial rapid decrease. This decrease in the energy charge recovers with increasing NAD + concentrations. The energy level correlates with the rates of incorporation of d, l-[4,5- 3H(N)]leucine into protein. ATP production via NAD + pyrophosphorylase or oxidative phosphorylation does not explain the stimulation by NAD +. Rather, the stimulation is correlated with the activation of glycolysis. Glycolysis is not active in lysate preparations because NAD + is absent. Additional possible roles of NAD + in protein synthesis are discussed.

O-methylhydroxylamine as a reagent for NAD + modification in urocanase

The inhibition of urocanase from Pseudomonas putida by O-methylhydroxylamine has been characterized as being due to the formation of an adduct between CH 3ONH 2 and NAD +, the latter of which has been recently shown to be a tightly bound coenzyme for this urocanase. Inhibition is maximal at pH 8.5 and is blocked by the presence of the substrate analog imidazole propionate. Loss of catalytic activity corresponds directly with the binding of 1 mol of 14CH 3ONH 2 per mole of enzyme, and partial reversibility of the modification, achieved by dialysis at pH 7.5, is accompanied by concomitant restoration of enzymatic activity. No incorporation of 14CH 3ONH 2 into urocanase is seen when enzyme-bound NAD + is first converted to NADH or when NAD + is removed by prior treatment of urocanase with 8 m urea. Stability and spectral properties of the CH 3ONH · NAD adduct are consistent with previous data reported for the product of the hydroxylamine reaction with NAD +. It is concluded that other urocanases which exhibit inhibition by hydroxylamine may likewise contain NAD + as an essential coenzyme and that the use of 14CH 3ONH 2 as a reversible modification reagent for NAD + should prove helpful for studies on the role of NAD + in the urocanase catalytic process.

Measurement of the oxidation-reduction potentials for two-electron and four-electron reduction of lipoamide dehydrogenase from pig heart.

The oxidation-reduction potential, E2, for the couple oxidized lipoamide dehydrogenase/2-electron reduced lipoamide dehydrogenase has been determined by measurement of equilibria of these enzyme species with lipoamide and dihydrolipoamide or with oxidized and reduced azine dyes. E2 is -0.280 V at pH 7, and deltaE2/deltapH is -0.06 V in the pH range 5.5 to 7.6. Values for E1, the oxidation-reduction potential for the couple 2-electron reduced enzyme/4-electron reduced enzyme, were obtained from measurements of the extent of dismutation of 2-electron reduced enzyme to form mixtures containing oxidized and 4-electron reduced enzyme. E1 is -0.346 V at pH 7, and deltaE1/deltapH is -0.06 V in the pH range 5.7 to 7.6. Spectra of oxidized enzyme and 4-electron reduced enzyme do not show variations with pH over this range, but the spectrum of the 2-electron reduced enzyme is pH-dependent, with the molar extinction at 530 nm changing from 3250 M-1 cm-1 at pH 8 to 2050 M-1 cm-1 at pH 5.2. The pH-dependent changes which are observed in the absorption properties of the 2-electron reduced enzyme are consistent with the disappearance of a charge transfer complex between an amino acid side chain and the oxidized flavin at the lower pH values, with the apparent pK of the side chain at pH 5. It has been suggested that the 530 nm absorbance of 2-electron reduced enzyme is due to a charge transfer complex between thiolate anion and oxidized flavin, and we propose that the thiolate anion is stabilized by interaction with a protonated base. The thermodynamic data predict that the amount of 4-electron reduced enzyme formed when the enzyme is reduced by excess NADH will be pH-dependent, with the greatest amounts seen at low pH values. These data support earlier evidence (Matthews, R.G., Wilkinson, K.D., Ballou, D,P., and Williams, C.H., Jr. (1976) in Flavins and Flavoproteins (Singer, T.P., ed) pp. 464-472; Elsevier Scientific Publishing Co., Amsterdam) that the role of NAD+ in the NADH-lipoamide reductase reaction catalyzed by lipoamide dehydrogenase is to prevent accumulation of inactive 4-electron reduced enzyme by simple reversal of the reduction of 2-electron reduced enzyme by NADH.

Response of nicotinamide adenine dinucleotide and cytochromes to aerobiosis and anaerobiosis on addition of glucose to saccharomyces cerevisiae

When aerated suspensions of whole yeast cells were either made anaerobic or were supplied with substrate (glucose), changes in absorption occurred which indicated cytochrome reduction. No such change was observed on addition of substrate to anaerobic suspension of yeast. These results were confirmed by obsevations of changes in absorbance at a series of fixed wavelengths corresponding to the reduced absorption peaks of cytochromesa, b andc. NAD* on the other hand was reduced on addition of substrate under both aerobic and anaerobic conditions. These results are discussed in relation to the possible role of NAD* in the Pasteur Effect.

Nicotinamide Adenine Dinucleotide As an Active Site Director in Glyceraldehyde 3-Phosphate Dehydrogenase Modification

Abstract Multivalent anions, sulfate, phosphate, or arsenate, selectively decrease the rate of alkylation of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase by both iodoacetamide and iodoacetate. N-Acetylation of the lysine residue that in the apoenzyme neighbors the essential cysteine residue does not eliminate this anion effect, suggesting that an alternative unique enzyme site is responsible for binding these anions. NAD+ (or 3-acetylpyridine adenine dinucleotide) at high concentration strikingly reverses this effect with iodoacetate; with iodoacetamide a further retardation in alkylation rate is observed. It is proposed that binding of NAD+ limits access of iodoacetamide or other uncharged reagents to the active site sulfhydryl group and simultaneously produces, via the pyridinium cation, a site capable of binding iodoacetate or any other negatively charged reagent. The role of NAD+ in acyl phosphatase and p-nitrophenyl hydrolase activities is considered in this context. The dependence on coenzyme concentration of the alkylation reaction for both reagents was determined with apoenzyme in various media. The results at low NAD+ concentrations in the presence of a multivalent anion indicate that a given coenzyme molecule facilitates, by means of the specific coulombic interaction, carboxymethylation (and not carbamylmethylation) at each of the sites to which it is bound during the reaction period. Finally the possible use of NAD+ (NADP+) as an active site director is commented upon in terms of modifying enzymatic behavior in vivo.

Function and role of NAD + in mechanism of action of rabbit-muscle glyceraldehydephosphate dehydrogenase

1. 1. NAD + is required for the oxidation of NADH by 1,3-diphosphoglycerate or acetyl phosphate. 2. 2. The maximal rate of acetyl phosphate reduction by NADH or APADH is reached when about 3 moles NAD + per mole enzyme are added. Larger amounts of NAD + inhibit with a K i (NADH as substrate) equal to 45 μM. 3. 3. The NAD-binding site with the lowest affinity for NAD + appears to be the most active in the dehydrogenase reaction. Under optimal conditions the E-(NAD) 3 complex is the catalytically active enzyme. 4. 4. The E-(NAD) 3 complex appears also to be the form with maximal catalytic activity in the transferase reactions. 5. 5. NAD + can be replaced by APAD + in both the dehydrogenase and arsenolysis reactions.