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Query: UNIPROT:P06889 (Mol)
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A series of Mud1 and Tn10 insertions were identified in the pncA chromosome region of Salmonella typhimurium which is responsible for the production of nicotinamide deamidase. Both pncA (resulting in no nicotinamide deamidase activity) and pncX (resulting in lowered nicotinamide deamidase activity) insertions were constructed. In addition, mutants which could utilize nicotinamide as a sole source of nitrogen were isolated. These mutants, designated pncH, hyperproduce nicotinamide deamidase. Genetic studies utilizing pncX--lacZ and pncA--lacZ operon fusions indicate that pncX::Tn10 insertions reduce transcription of pncA--lac while pncH mutations increase the expression of both pncA--lacZ and pncX-lacZ. The gene order was determined as purB--pncA--pncX--gdh with transcription of both pncA and pncX occurring in the counterclockwise direction. Merodiploid studies suggest a model whereby pncX and pncA form an operon with the major promoter occurring upstream from pncX. A second, weaker promoter for pncA must be situated between pncX and pncA. The pncH mutations appear to occur in the pncX promoter (pncXp) increasing promoter activity.
Mol Gen Genet 1986 Dec
PMID:The pyridine nucleotide cycle of Salmonella typhimurium: genetic characterization of the pncXA operon. 355 Mar 86

The 31P NMR spectrum of the adult tapeworm, Hymenolepis diminuta, at 37 degrees C during perfusion with physiological saline was composed of 10 peaks. Based on chemical shifts and analysis of worm extracts, the phosphorus components included glucose-6-phosphate, fructose-6-phosphate, phosphorylcholine, glycerophosphoryl choline and -ethanolamine, nucleotide monophosphate-diphosphate and -triphosphate, nicotinamide adenine dinucleotide and uridine diphosphate glucose. The mean level of nucleotide triphosphate was 0.86 nmol (mg fresh weight)-1 and the nucleotide triphosphate/-diphosphate ratio 3.9. Based on the nucleotide triphosphate level, worms were viable for at least 3 h and the intracellular pH was maintained constant at approximately 6.7. Short-term exposure to mebendazole perfused at 11 or 27 microM solubilized in physiological saline containing 0.5% Tween 80 or 0.1% dimethyl sulphoxide had little effect on the nucleotide triphosphate level. Some cytological changes, however, were evident following perfusion of mebendazole. In contrast, exposure to 2,4-dinitrophenol caused a rapid decline in nucleotide triphosphate level. It was concluded that mebendazole does not exert its primary effect on oxidative phosphorylation.
Mol Biochem Parasitol 1987 Jan 02
PMID:In vivo 31P NMR spectrum of Hymenolepis diminuta and its change on short-term exposure to mebendazole. 380 50

We prepared primary monolayer cultures of adult rat hepatocytes and measured the losses of cytochromes P-450 with the use of specific antibodies directed against purified forms of hepatic cytochrome P-450 which predominate in untreated rats (P-450UT-A, P-450UT-F) or in rats treated with phenobarbital (P-450PB-B/D, P-450PB-C, P-450PB/PCN-E) or with 3-methylcholanthrene (P-450 beta NF-B, P-450 beta NF/ISF-G). In hepatocytes prepared from an untreated rat and incubated in control medium, total cytochrome P-450, measured spectrally as CO-binding hemoprotein, declined 68% during the first 72 hr in culture. However, the sum of the immunoreactive cytochromes P-450 declined only 24%, indicating that loss of heme rather than of protein accounts for much of the well-known loss of cytochromes P-450 in hepatocyte cultures. In cultures prepared from untreated rats or from rats treated with phenobarbital or with 3-methylcholanthrene, individual forms of cytochrome P-450 declined at markedly differing rates. Incubation of cultures in three different media previously reported to maintain levels of total cytochrome P-450 failed to prevent the decline in total cytochrome P-450 during the first 24 to 72 hr in culture. However, in cultures incubated in medium containing metyrapone, the level of holocytochrome P-450 was maintained at the initial value during the first 72 hr, apparently by preventing the net loss of cytochrome P-450 heme and by increasing the concentrations of immunoreactive P-450PB/PCN-E and P-450 beta NF-B. Medium containing nicotinamide increased the proportion of P-450 beta NF-B relative to the other forms of cytochrome P-450, whereas cysteine-free medium increased P-450UT-F. We conclude that loss of cytochrome P-450 in cultured hepatocytes involves loss of its heme moiety coupled with changes in the concentrations of the individual forms. Recognition of these changes as influenced by specific components of the culture medium is important when using primary hepatocyte cultures for study of xenobiotic metabolism and toxicity in the liver.
Mol Pharmacol 1985 Jan
PMID:Changes in the concentration of seven forms of cytochrome P-450 in primary cultures of adult rat hepatocytes. 396 24

Rates of NADPH production via the pentose phosphate cycle were determined in perfused livers from phenobarbital-treated rats by measuring 14CO2 production from [1-14C]glucose infused in the presence and absence of p-nitroanisole (0.2 mM), a substrate for mixed-function oxidation. In the fed state, basal rates of NADPH generation were 34-44 mumol/g/hr. p-Nitroanisole, which was metabolized at rates of 8.9 mumol/g/hr, stimulated pentose cycle-dependent NADPH production by 21-24 mumol/g/hr. Fasting for 24 hr prior to perfusion diminished pentose cycle flux by 80% and largely abolished the stimulation of the pentose cycle by p-nitroanisole. In contrast, rates of p-nitroanisole O-demethylation were only diminished slightly, to 5.7 mumol/g/hr. Fasting decreased hepatic glucose, glucose 6-phosphate, and 6-phosphogluconate contents drastically as expected. Pretreatment of rats with 6-aminonicotinamide, which is metabolized to a potent inhibitor of 6-phosphogluconate dehydrogenase, decreased rates of NADPH generation via the pentose cycle to 6.9 mumol/g/hr but did not alter rates of p-nitroanisole metabolism (8.8 mumol/g/hr). Basal rates of NADPH generation decreased from 38 to 26 mumol/g/hr during infusion of potassium cyanide (2 mM), an inhibitor of mitochondrial energy metabolism. Cyanide also decreased rates of p-nitroanisole O-demethylation by over 60%; however, stimulation of NADPH generation via the pentose cycle by p-nitroanisole was as great in the presence (17-21 mumol/g/hr) as in the absence of cyanide. Since rates of mixed-function oxidation were unaffected after virtually complete inhibition of the pentose cycle with 6-amino-nicotinamide, it is concluded that reducing equivalents for the mixed-function oxidation of p-nitroanisole are not provided by the pentose cycle under these conditions.
Mol Pharmacol 1985 Oct
PMID:Rates of pentose cycle flux in perfused rat liver. Evaluation of the role of reducing equivalents from the pentose cycle for mixed-function oxidation. 405 19

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.
Mol Cell Biol 1981 Feb
PMID:Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism. 615 51

The recently characterized amino acid L-arogenate (Zamir et al., J. Am. Chem. Soc. 102:4499-4504, 1980) may be a precursor of either L-phenylalanine or L-tyrosine in nature. Euglena gracilis is the first example of an organism that uses L-arogenate as the sole precursor of both L-tyrosine and L-phenylalanine, thereby creating a pathway in which L-arogenate rather than prephenate becomes the metabolic branch point. E. gracilis ATCC 12796 was cultured in the light under myxotrophic conditions and harvested in late exponential phase before extract preparation for enzymological assays. Arogenate dehydrogenase was dependent upon nicotinamide adenine dinucleotide phosphate for activity. L-Tyrosine inhibited activity effectively with kinetics that were competitive with respect to L-arogenate and noncompetitive with respect to nicotinamide adenine dinucleotide phosphate. The possible inhibition of arogenate dehydratase by L-phenylalanine has not yet been determined. Beyond the latter uncertainty, the overall regulation of aromatic biosynthesis was studied through the characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and chorismate mutase. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase was subject to noncompetitive inhibition by L-tyrosine with respect to either of the two substrates. Chorismate mutase was feedback inhibited with equal effectiveness by either L-tyrosine or L-phenylalanine. L-Tryptophan activated activity of chorismate mutase, a pH-dependent effect in which increased activation was dramatic above pH 7.8 L-Arogenate did not affect activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase or of chorismate mutase. Four species of prephenate aminotransferase activity were separated after ion-exchange chromatography. One aminotransferase exhibited a narrow range of substrate specificity, recognizing only the combination of L-glutamate with prephenate, phenylpyruvate, or 4-hydroxyphenylpyruvate. Possible natural relationships between Euglena spp. and fungi previously considered in the literature are discussed in terms of data currently available to define enzymological variation in the shikimate pathway.
Mol Cell Biol 1981 May
PMID:The aromatic amino acid pathway branches at L-arogenate in Euglena gracilis. 615 55

NAD glycohydrolase activity was studied using bovine erythrocytes, erythrocyte ghosts and partially purified enzyme preparations. During catalysis the enzyme becomes irreversibly inactivated in a process related to substrate turnover. Self-inactivation was observed with intact cells, ghosts and solubilized enzyme and could be demonstrated with NAD, NADP and nicotinamide 1,N6 ethenoadenine dinucleotide as substrates. Thionicotinamide adenine dinucleotide and NADH, which are not substrates for the enzyme, do not inactivate but are reversible substrate-competitive inhibitors. Added thiols had no effect on enzyme self-inactivation. Of the reaction products, added nicotinamide partially protected the enzyme while added ADPR had no effect. Thermodynamic parameters calculated from Arrhenius plots for rate constants of self-inactivation indicate a large negative delta S for transition state formation suggesting a process other than extensive denaturation. Erythrocyte ghost NADases from several other mammalian sources have been demonstrated to undergo a self-inactivation similar to that observed with the bovine enzyme.
Mol Cell Biochem 1980 May 28
PMID:Self-inactivation of an erythrocyte NAD glycohydrolase. 624 64

The conformation of NAD+ in the sheep liver sorbitol dehydrogenase-NAD+ binary complex has been investigated using time-dependent proton-proton transferred nuclear Overhauser enhancement measurements to determine interproton distance ratios and distances between bound NAD+ protons. The conformation about both the adenosine and nicotinamide riboside glycosidic bonds is anti, the conformations of the adenosine and nicotinamide ribose rings are C3'-endo and C1'-exo, respectively, and the conformations about the adenosine and nicotinamide riboside C4'-C5' bonds are g+ and t, respectively, similar to those found in complexes of NAD+ with other A type dehydrogenases. In addition, however, the distance data are indicative of an unusual overall conformation of NAD+ in the sorbitol dehydrogenase-NAD+ binary complex, with the planes of the nicotinamide and adenine rings separated by 6 to 8 A and at approximately 120 degrees to each other. This overall conformation differs from the concensus extended conformation found in the NAD+-dehydrogenase complexes crystallized to date, where the planes of the nicotinamide and adenine rings are 12 to 14 A apart and nearly perpendicular to each other.
J Mol Biol 1984 Feb 05
PMID:An unusual conformation of NAD+ bound to sorbitol dehydrogenase? A time-dependent transferred nuclear Overhauser effect study. 631 20

Rat muscle glyceraldehyde-3-phosphate dehydrogenase is one of several enzymes which have been found to undergo age-related modifications. While the amount of this enzyme in muscle tissue does not change with age, both its specific activity and affinity towards its co-enzyme are significantly reduced in the old tissue. Age-related structural changes were found to exist in the nicotinamide binding site of the enzyme and the reactions leading to the activity loss in 'old' glyceraldehyde-3-phosphate dehydrogenase were shown to involve a reversible modification of the essential cysteine-149 residue at the active site of the enzyme. The aging effects were simulated by a controlled oxidation of cys-149 in samples of 'young' glyceraldehyde-3-phosphate dehydrogenase and subsequent reduction of this residue by 2-mercaptoethanol. The enzyme modified in this way closely resembles native 'old' glyceraldehyde-3-phosphate dehydrogenase, indicating that the structural modifications in the latter enzyme are indeed introduced by a post-translational process. The mechanism for aging of glyceraldehyde-3-phosphate dehydrogenase which is proposed, based on these observations, thus assumes an oxidation of cys-149 as its first step followed by irreversible conformational changes in the enzyme molecule. The aging of glyceraldehyde-3-phosphate dehydrogenase may thus be triggered by the reduced ability of old muscle tissue to protect its constituents against oxidation.
Mol Cell Biochem 1984
PMID:Age-related effects in enzyme catalysis. 636 9

The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.
J Mol Biol 1983 Oct 25
PMID:Comparison of AMP and NADH binding to glycogen phosphorylase b. 641 89


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