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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the delta 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-14C]eicosa-8,11,14-trienoic acid (20:3n-6), was separated from the product [1-14C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The delta 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 microM. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of delta 5 desaturase were 56 microM and 60 pmoles.min-1.mg-1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the delta 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.
Mol Cell Biochem 1993 Dec 08
PMID:Delta 5 desaturase activity in rat kidney microsomes. 817 24

We have solved the structure of the binary complex of the glutamate dehydrogenase from Clostridium symbiosum with glutamate to 1.9 A resolution. In this complex, the glutamate side-chain lies in a pocket on the enzyme surface and a key determinant of the enzymic specificity is an interaction of the substrate gamma-carboxyl group with the amino group of Lys89. In the apo-enzyme, Lys113 from the catalytic domain forms an important hydrogen bond to Asn373, in the NAD(+)-binding domain. On glutamate binding, the side-chain of this lysine undergoes a significant movement in order to optimize its hydrogen bonding to the alpha-carboxyl group of the substrate. Despite this shift, the interaction between Lys113 and Asn373 is maintained by a large-scale conformational change that closes the cleft between the two domains. Modelling studies indicate that in this "closed" conformation the C-4 of the nicotinamide ring and the alpha-carbon atom of the amino acid substrate are poised for efficient hydride transfer. Examination of the structure has led to a proposal for the catalytic activity of the enzyme, which involves Asp165 as a general base, and an enzyme-bound water molecule, hydrogen-bonded to an uncharged lysine residue, Lys125, as an attacking nucleophile in the reaction.
J Mol Biol 1993 Dec 20
PMID:Conformational flexibility in glutamate dehydrogenase. Role of water in substrate recognition and catalysis. 826 17

We have analysed the sequence homology between glutamate, leucine and phenylalanine dehydrogenases in the light of the solution of the structure of the glutamate dehydrogenase from Clostridium symbiosum. This analysis indicates that the elements of secondary structure comprising the core of the two domains in glutamate dehydrogenase are conserved in the other two enzymes. There is a striking conservation of the residues responsible for the recognition of the nicotinamide ring of the nucleotide cofactor and the backbone of the amino acid substrates. Furthermore, residues involved in a major conformational rearrangement on amino acid binding are preserved, as are those implicated in the catalytic chemistry. In contrast, the pattern of insertions/deletions between these enzymes is consistent with possible differences in quaternary structure. Differential substrate specificity between these enzymes is achieved by critical substitutions at the base of the binding pocket, which accommodates the side-chain of the amino acid substrate. This provides insights into the mutations necessary to produce new catalysts for the chiral synthesis of novel amino acids.
J Mol Biol 1993 Dec 20
PMID:Evolution of substrate diversity in the superfamily of amino acid dehydrogenases. Prospects for rational chiral synthesis. 826 39

The role of Ca(2+)-stimulated adenosine 5'-triphosphatase (Ca(2+)-ATPase) in Ca2+ sequestering of rat liver nuclei was investigated. Ca(2+)-ATPase activity was calculated by subtracting Mg(2+)-ATPase activity from (Ca(2+)-Mg2+)-ATPase activity. Ca2+ uptake and release were determined with a Ca2+ electrode. Nuclear Ca(2+)-ATPase activity increased linearly in the range of 10-40 microM Ca2+ addition. With those concentrations, Ca2+ was completely taken up by the nuclei dependently on ATP (2 mM). Nuclear Ca(2+)-ATPase activity was decreased significantly by the presence of arachidonic acid (25 and 50 microM), nicotinamide-adenine dinucleotide (NAD+; 2 mM) and zinc sulfate (2.5 and 5.0 microM). These reagents caused a significant decrease in the nuclear Ca2+ uptake and a corresponding elevation in Ca2+ release from the nuclei. Moreover, calmodulin (10 micrograms/ml) increased significantly nuclear Ca(2+)-ATPase activity, and this increase was not seen in the presence of trifluoperazine (10 microM), an antagonist of calmodulin. The present findings suggest that Ca(2+)-ATPase plays a role in Ca2+ sequestering by rat liver nuclei, and that calmodulin is an activator. Moreover, the inhibition of Ca(2+)-ATPase may evoke Ca2+ release from the Ca(2+)-loaded nuclei.
Mol Cell Biochem 1993 Aug 11
PMID:Characterization of Ca(2+)-stimulated adenosine 5'-triphosphatase and Ca2+ sequestering in rat liver nuclei. 826 71

The effect of nicotinamide-adenine dinucleotides (NAD+ and NADP+) on Ca2+ transport in rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. Ca2+ uptake was dependent on adenosine triphosphate (ATP; 2 mM). The presence of NAD+ (2 mM) or NADP+ (1 and 2 mM) caused a significant inhibition of Ca2+ uptake following addition of 2 mM ATP. Ca2+, which accumulated in the nuclei during 6 min after ATP addition, was significantly released by the addition of NAD+ (0.5-2 mM) or NADP+ (0.1-2 mM). However, the effect of NADH (2 mM) or NADPH (2 mM) on Ca2+ uptake and release clearly weakened in comparison with the effects of NAD+ and NADP+. Meanwhile, ryanodine (10 microM), thapsigargin (10 microM) or oxalate (0.5 mM) had no effect on Ca2+ uptake and release in rat liver nuclei. These reagents did not significantly alter the effects of 2 mM NAD+ on Ca2+ uptake and release. Thus, NAD+ and NADP+ had a potent effect on Ca2+ transport in rat liver nuclei. The present findings suggest that the liver cytosolic NAD+ (NADP+) is a factor in the regulation of the nuclear Ca2+ concentration.
Mol Cell Biochem 1993 Apr 21
PMID:Effect of nicotinamide-adenine dinucleotides on Ca2+ transport system in rat liver nuclei: stimulation of Ca2+ release by NAD+. 831 29

The structure of malate dehydrogenase from Escherichia coli complexed with the substrate analog, citrate and the cofactor NAD, has been determined by X-ray crystallography. A monoclinic crystal of the malate dehydrogenase, grown in citrate buffer, was soaked in 10 mM NAD solution and found to be isomorphous with the apo-form. The X-ray data extended to 1.9 A, nearly the same resolution limit as the apo-enzyme crystals. The ternary complex of malate dehydrogenase has very few conformational differences from that of the pseudo binary complex of enzyme with bound citrate. In addition, the NAD molecule has a very similar conformation to the NAD as found in the crystal structure of the cytosolic eukaryotic malate dehydrogenase. Similar hydrogen bond interactions are made by both enzymes from polar groups belonging to the NAD. Such interactions include hydrogen bonds from the ribose oxygens and the phosphate oxygens, to backbone amide and carbonyl atoms of the protein and to side-chains of a select few conserved hydrophilic residues. The only notable difference occurs in the active site region where the nicotinamide moiety is obstructed from further entering the active site by the C-6 carbonyl atoms of citrate. In this position there are no direct polar interactions between the protein and the nicotinamide moiety. Energy minimization of the structure with malate substituted for citrate in the active site shows that the nicotinamide moiety assumes the same position in the active site as the NAD in cytosolic malate dehydrogenase. The carboxamide atoms of the energy minimized model make significant hydrogen bond interactions with the catalytic residue, H177, and with the main-chain atoms of I117 and V146 in the vicinity of the active site, while the position of the rest of the cofactor remains unchanged.
J Mol Biol 1993 Jul 05
PMID:Crystal structure of a ternary complex of Escherichia coli malate dehydrogenase citrate and NAD at 1.9 A resolution. 833 58

Incubation of pituitary GH1 cells with N'-methylnicotinamide, nicotinamide and 3-acetylpyridine which inhibit nuclear ADP-ribosylation and/or the cellular concentration of its substrate NAD+ reduced the amount of nuclear thyroid hormone receptors in a time- and dose-dependent manner without altering the affinity of the receptors for the hormone. A transient activation of poly(ADP-ribose)polymerase by methyl methanesulfonate, ultraviolet irradiation or spermine caused a rapid depletion of cellular NAD+ content and was followed by a strong inhibition of ADP-ribosylation. These agents also produced a very rapid and marked reduction of receptor numbers. The decrease of receptors caused by the different compounds is not secondary to a generalized inhibition of protein synthesis or to an alteration in hormone availability. The abundance of c-erbA alpha and beta mRNAs, which encode thyroid hormone receptors, was reduced in cells treated with the compounds that decrease receptor number, thus suggesting that this effect is caused by a decrease in the expression of c-erbA genes.
Mol Cell Endocrinol 1993 Feb
PMID:Nicotinamide analogs and DNA-damaging agents deplete thyroid hormone receptor and c-erbA mRNA levels in pituitary GH1 cells. 838 10

Purified rat pancreatic insulin-producing B-cells, which display a 12-fold higher activity of FAD-linked glycerophosphate dehydrogenase than other islet endocrine cells, were exposed for 30 min to 2 mM streptozotocin and subsequently cultured for 2 days in the absence or presence of 2 mM nicotinamide. Streptozotocin decreased by 54% the number of B-cells and, in surviving cells, lowered by 75% the activity of FAD-linked glycerophosphate dehydrogenase, whilst failing to affect that of glutamate dehydrogenase. This coincided with a 42-51% reduction of insulin secretion, when expressed relative to either the DNA or hormonal content of surviving cells. After exposure to streptozotocin, the presence of nicotinamide in the culture medium reduced cell death by 44% and also reduced the deleterious effects of streptozotocin upon both the enzymic and secretory activities of surviving cells. These findings indicate that the decreased activity of FAD-linked glycerophosphate dehydrogenase previously documented in pancreatic islets from streptozotocin-injected rats, as well as the protective effect of nicotinamide thereupon, are not attributable solely to changes in the number of B-cells but also to an altered enzymic activity in surviving B-cells. The latter anomaly may account, in part at least, for an impaired B-cell secretory response to D-glucose.
Mol Cell Biochem 1993 Mar 24
PMID:Effect of streptozotocin and nicotinamide upon FAD-glycerophosphate dehydrogenase activity and insulin release in purified pancreatic B-cells. 848 53

We report the frequent occurrence in proteins of motifs consisting of either 9-membered or 11-membered rings that involve the side-chain amide groups of asparagine and glutamine residues. The syn CO and NH groups of these amide groups are hydrogen-bonded to the main-chain NH and CO groups of other amino acid residues. The main-chain part of both the 9-membered and 11-membered rings has the conformation of a beta-strand. One such ring motifs occurs, on average, in half of all the proteins we examined. Similar conformations are found for most examples of the 9-membered and 11-membered rings. One of the 11-membered rings is distinct, compared to the others, in that its main-chain part has a mirror-image conformation. Another of the 11-membered rings occurs at the interior of the variable domains of some antibodies and assists in linking the two beta-sheets. We observe one 9-membered ring structure in a dihydrofolate reductase complex in which the amide in the nicotinamide group of the ligand NADP is bound to the enzyme. Groups that can form hydrogen bonds in a similar way to amide groups occur in several nucleotide bases; we find one example of a 9-membered ring involving adenine and main-chain atoms in the FAD-protein complex of glutathione reductase. Both have conformations like those of the other 9-membered rings.
J Mol Biol 1993 Jun 05
PMID:Common ring motifs in proteins involving asparagine or glutamine amide groups hydrogen-bonded to main-chain atoms. 851 58

In a previous report we described that adenosine-induced apoptosis of HL-60 cells was blocked by the pretreatment of cells with a potent inhibitor (3-aminobenzamide) of poly(ADP-ribose) polymerase (PARP). The pretreatment of the cells with nicotinamide, another inhibitor of the enzyme, also suppressed most effectively the adenosine-induced apoptosis. This inhibition was reversible and observed during apoptosis mediated by other known apoptosis inducers such as actinomycin D and staurosporine (group I inducers), but nicotinamide was ineffective on the apoptosis mediated by VM 26, camptothecin and A23187 (group II inhibitors). In addition to the enzyme inhibition, a down-regulation of the enzyme level caused by the pretreatments of cells with differentiation-inducing agents, retinoic acid (RA) and dimethylsulfoxide (DMSO) also resulted in a marked resistance of the cells to the apoptosis inducers. A pretreatment of the cells for a limited time of 24 hrs. by these agents decreased the PARP level to 66-75% of the untreated cells and the cells showed a quite similar resistance to the group I apoptosis inducers like the cells treated with the enzyme inhibitors, whereas they were still sensitive to the group II inhibitors. A more prolonged treatment for 48 hrs. of the cells with RA and DMSO resulted in further down-regulation of the cellular PARP reaching respectively 50 and 43% of control cells and at this stage, the cells became resistant to all the inducers of both groups. These results suggest that the pathway, by which both groups of the inducers initiate and progress apoptosis, is not identical but include at least two different processes which are differently affected by PARP-inhibition or by different levels of cellular PARP.
Cell Mol Biol (Noisy-le-grand) 1995 Sep
PMID:Inhibition and down-regulation of poly(ADP-ribose) polymerase results in a marked resistance of HL-60 cells to various apoptosis-inducers. 853 70


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