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At least 19 recordsLinked to original sources
PubMed1976

Influence of dilution rate on NAD(P) and NAD(P)H concentrations and ratios in a Pseudomonas sp. grown in continuous culture.

A freshwater Pseudomonas sp. was grown in continuous culture under steady-state conditions in L-lactate-, succinate-, glucose- or ammonium-limited media. Under carbon limitation, the NAD(H) (i.e. NAD + NADH) concentration of the organisms increased exponentially from approximately 2 to 7 mumol/g dry wt as the culture dilution rate (D) was decreased from 0.5 to 0.02 h-1. Organisms grown at a given D in any of the carbon-limited media possessed very similar levels of NAD(H). Therefore, under these conditions, cellular NAD(H) was only a function of the culture O and was independent of the nature of the culture carbon source. D had no influence on the NAD(H) content of cells grown under ammonium limitation. In contrast, cellular NADH concentration was not influenced by D in carbon- or ammonium-limited media. In L-lactate-limited medium, bacteria possessed 0.14 mumol NADH/g dry wt; very similar levels were found in organisms grown in the other media. The results are consistent with those of Wimpenny & Firth (1972) that bacteria rigidly maintain a constant NADH level rather than a constant constant NADH: NAD ratio. NADP(H) (i.e. NADP + NADPH) and NADPH levels were also not influenced by changes in the culture carbon source or in D; in L-lactate-limited medium these concentrations were 0.97 and 0.53 mumol/g cell dry wt, respectively. The NADPH:NADP(H) ratio was much higher than the NADH:NAD(H) ratio, averaging 55% in carbon-limited cells.

Culture Media↗
PubMed1976

8-Azidoacenine analogs of NAD+ and FAD. Synthesis and coenzyme properties with NAD+-dependent and FAD-dependent enzymes.

The synthesis and purification of the 8-azidoadenine analogs of NAD+ (azido-NAD+) and FAD (AZIDO-FAD) from 8-azidoadenosine 5'-phosphate and NMN+ or FMN, respectively, is described. The coenzyme analogs are characterized by absorption, nuclear magnetic resonance and circular dichroism spectra. The two latter methods indicate a folded structure of azido-NAD+ and azido-FAD. Upon irradiation at 300 mn in aqueous solution, a change of the ultraviolet absorption spectra of the coenzyme analogs indicates photolysis of the azido group. The coenzyme properties of azido-NAD+ are demonstrated with lactate, glutamate and alcohol dehydrogenase yielding 14, 154 and 60%, respectively, of the V observed with NAD+. Concomitantly, the Km values of the coenzyme analogs are 1.7, 3.5 and 3-fold higher than those of NAD+. Azido-FAD is shown to be coenzyme of apo-glucose oxidase. The recovery of activity, however, is much slower in the presence of azido-FAD than with FAD. A final value of 66% of the activity with FAD is obtained. With apo-D-amino acid oxidase, azido-FAD is completely inactive, although it is specifically bound to the enzyme.

Alcohol Oxidoreductases↗
PubMed1977

Spectrophotometric studies on NAD(P)H oxidase of leukocytes. 1. The relationship between granule-NAD(P)H oxidase and myeloperoxidase.

The NAD(P)H oxidase located in granules from resting leukocytes seems to be identical with myeloperoxidase on the basis of the following results. Spectral changes representing the difference between granules with and without NAD(P)H under various conditions represented the formation of compound III of myeloperoxidase, corresponding to the oxidation of NAD(P)H. The KCN difference spectrum of granules from both resting and phagocytizing leukocytes was in agreement with the KCN difference spectrum of myeloperoxidase. The affinity of KCN for myeloperoxidase was the same in both resting and phagocytizing leukocytes. The KCN-sensitive portion of NAD(P)H oxidase of granules from phagocytizing leukocytes seems to be identical with isolated myeloperoxidase and the myeloperoxidase of resting leukocytes. The KCN-insensitive oxidation of NAD(P)H by granules from phagocytizing leukocytes has not been found to be identical with myeloperoxidase.

Animals↗
PubMedDate not supplied

Synthesis and degradation of NAD in guinea pig cardiac muscle: I. Dependence upon the extracellular concentration of nicotinamide and nicotinic acid.

The NAD concentration as well as the 14C-incorporation in NAD and the disappearance of 14C-NAD were studied in spontaneously beating atria of guinea pigs at high and low concentrations of the precursors nicotinamide or nicotinic acid. Atria were incubated in Krebs-Henseleit solution containing 15 mM glucose and the appropriate precursors at 30 degrees C. The control NAD concentration (33 nMol/100 mg w.w.) remained unchanged during a 24-h-incubation time. -20 mM 14C-nicotinamide increased the total NAD about three-fold (90 nMol/100 mg w.w.) after an incubation period of 24 h, with positive effects on the performance. The incorporation rate in 14C-NAD was calculated to be 43.7 nMol/100 mg w.w. . 24 h. The ADPR moiety for the NAD synthesis stemmed from an endogenous pool. Between 5 and 20 mM nicotinamide the increase in the NAD concentration followed an apparent Michaelis-Menten kinetics with a Km of 6.1 mM nicotinamide and a Vmax of 70.92 nMol NAD/100 mg w.w. . 24 h. This can be explained as a new synthesis of NAD by a high concentration of nictoinamide and also by a decreased degradation of NAD, due to inhibition of the glycohydrolase by the high concentration of nicotinamide. The ratio of incorporation and disappearance of 14C-NAD during the 8th and 16th h incubation period was 2:1. After pre-incubation with 20 mM nicotinamide for an 8-h period the NAD concentration decreased to normal values after incubation for 8 h in a nicotinamide free medium. -20 mM 14C-nicotinic acid did not change the total NAD level and no significant incorporation in 14C-NAD could be detected, whereas negative effects on the performance occurred. -10 muM 14C-nicotinamide showed a slight increase in the total NAD concentration (39.7 nMol/100 mg w.w.) and in the 14C-incorporation (4.8 nMol/100 mg w.w.) within 24 h. -10 muM 14C-nicotinic acid seemed here to be the better precursor in this concentration. The NAD concentration increased to 49.8 nMol/100 mg w.w. after a 16 h incubation period and the incorporation in 14C-NAD was 12.1 nMol/100 mg w.w. after an incubation time of 24 h. As consequences of the observed different influences of each precursor on NAD turnover and NAD concentration the pathways of NAD synthesis and degradation must be studied. The importance of an increased NAD level for the energy metabolism of the cardiac muscle under aerobic and anaerobic conditions is discussed.

Animals↗
PubMed1977

Studies on NAD+ permeability in intact mitochondria from rabbit reticulocytes.

Functionally intact mitochondria from rabbit reticulocytes are characterized by a low NAD+ level after the preparation (0.29 nmoles NAD+ + NADH/mg protein). They are apparently impermeable for NADH and exhibit a slow net uptake of NAD+. From the increase of O2-uptake in state 3 and the increase of NADH concentration in state 4 of respiration after the addition of NAD+ we concluded that 3--10 min are necessary for the saturation with NAD+ at 23 degrees C. 2mM NAD+ extramitochondrially are not sufficient to saturate the mitochondria with NADH and probably NAD+, too. Because of the net uptake of NAD+ we assume that reticulocyte mitochondria lose NAD+ during their preparation. If they are incubated with the physiological concentration of 300 micrometer NAD+, which was found in reticulocytes, a value of 1.9 nmoles NAD+ + NADH mg protein was calculated. At an extramitochondrial NAD+ concentration of 300 micrometer, reticulocyte mitochondria exhibit an almost maximal O2-uptake in the presence of oxaloacetate or alpha-ketoglutarate. It is concluded that the mitochondria in intact reticulocytes contain the "normal" complement of NAD+ + NADH.

Animals↗
PubMed1975

Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD.

Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results establish that in the respiratory chain of beef-heart mitochondria NADH oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are independent reactions.

Animals↗
PubMed1979

Subunit interactions in rabbit-muscle glyceraldehyde-phosphate dehydrogenase, as measured by NAD+ and NADH binding.

1. The binding parameters for NADH and NAD+ to rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been measured by quenching of the flourescence of the protein and the NADH. 2. The fact that the degree of protein fluorescence quenching by bound NAD+ or NADH, excited at 285 nm and measured at 340 nm ('blue' tryptophans), is not linearly related to the saturation functions of these nucleotides, leads to a slight overestimation of the interaction energy and an underestimation of the concentration of sites, if linearity is assumed. 3. This is also the case for NADH, but not for NAD+, when the protein fluorescence is excited at 305 nm and measured at 390 nm ('red' tryptophans). 4. The binding of NAD+ can be described by a model in which the binding of NAD+, via negative interactions within the dimer, induces weaker binding sites, with the result that the microscopic dissociation constant is 0.08 microM at low saturation and 0.18 microM for the holoenzyme. 5. The binding of NADH can be described on the basis of the same model, the dissociation constant at low saturation being 0.5 microM and of the holoenzyme 1.0 microM. 6. The fluorescence of bound NADH is not sensitive to the conformational changes that cause the decrease in affinity of bound NAD+ or NADH. 7. The binding of NAD+ to the 3-phosphoglyceroyl enzyme can be described by a dissociation constant that is at least two orders of magnitude greater than the dissociation constants of the unacylated enzyme. The affinity of NAD+ to this form of the enzyme is in agreement with the Ki calculated from product inhibition by NAD+ of the reductive dephosphorylation of 1,3-diphosphoglycerate.

Acylation↗
PubMed1978

NAD in muscle of man at rest and during exercise.

NAD can be used to assess the adequacy of oxygen availability to the respiratory chain. An enzymatic assay was established for NAD in human muscle biopsy samples. It gave reliable, reproducible results. The variation within and between subjects was less than 12%. Muscle NAD and lactate were determined at rest, and after bicycle ergometry work requiring approximately 75 and approximately 100% VO2 max (six subjects, four tests each). A positive (P less than 0.01) linear relationship between resting muscle NAD and percent slow twitch fibers was found, suggesting that fiber types may have different NAD content. Muscle NAD decreased during submaximal and maximal work (P less than 0.05). A large portion (73%) of the NAD reduction could be accounted for by increased muscle water. No relationship could be established between NAD and lactate. The negative linear relationship (P less than 0.01) between the muscle/blood ratio and percent slow twitch fibers is another indication of the fiber having different metabolic responses to the activity.

Adult↗