Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase

Bredt, David S.; Hwang, Paul M.; Glatt, Charles E.; Lowenstein, Charles J.; Reed, Randall R.; Snyder, Solomon H.

doi:10.1038/351714a0

Cited by 2,257 publications

(584 citation statements)

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“…NOS was originally divided into two functional classes based on sensitivity to calcium, but differences in properties and differential inhibitor/ substrate specificity suggest that there are three major forms of NOS. This has recently been confirmed by the cloning and characterization of three distinct NOS cDNAs [1][2][3]. This permits the definitive classification of the gene products so far characterized in this way as neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) [4].…”

Section: Introductionmentioning

confidence: 89%

Up‐regulation of nitric oxide synthase by estradiol in human aorticendothelial cells

et al. 1995

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We have examined the effects of sex hormones on calcinm-dependent NO production and protein levels of NO synthase in cultured human aortic endothelial cells, which were treated with various doses of 17~-estradiol and testosterone for 8-48 h. Treatment with 17~-estradiol enhanced calcium-dependent NO production, but testosterone had exerted no effect. Western blot using monoclonal anti-human endothelial NO synthase antibody clarified that increased NO production by 17~-estradiol treatment was accompanied by increased NO synthase protein.Our results provide evidence that human endothelial NO synthase can be regulated by estrogens.

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Section: Introductionmentioning

confidence: 89%

Up‐regulation of nitric oxide synthase by estradiol in human aorticendothelial cells

et al. 1995

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“…The N-terminal oxygenase domain containing heme, BH 4 , and L-arginine binding sites is a P450-type hemoprotein, but does not show sequence homology to other known P450s. A CaM binding module exists near the center of the NOS sequences (9), and binding of Ca 2ϩ /CaM facilitates electron transfer from the reductase to the oxygenase domain (10,11).…”

mentioning

confidence: 99%

Mutation of Glu-361 in Human Endothelial Nitric-oxide Synthase Selectively Abolishes L-Arginine Binding without Perturbing the Behavior of Heme and Other Redox Centers

Chen

Tsai

Berka

et al. 1997

Journal of Biological Chemistry

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Nitric oxide (NO) and L-citrulline are formed from the oxidation of L-arginine by three different isoforms of NO synthase (NOS). Defining amino acid residues responsible for L-arginine binding and oxidation is a primary step toward a detailed understanding of the NOS reaction mechanisms and designing strategies for the selective inhibition of the individual isoform. We have altered Glu-361 in human endothelial NOS to Gln or Leu by site-directed mutagenesis and found that these mutations resulted in a complete loss of L-citrulline formation without disruption of the cytochrome c reductase and NADPH oxidase activities. Optical and EPR spectroscopic studies demonstrated that the Glu-361 mutants had similar spectra either in resting state or reduced CO-complex as the wild type. The heme ligand, imidazole, could induce a low spin state in both wild-type and Glu-361 mutants. However, unlike the wild-type enzyme, the low spin imidazole complex of Glu-361 mutants was not reversed to a high spin state by addition of either L-arginine, acetylguanidine, or 2-aminothiazole. Direct L-arginine binding could not be detected in the mutants either. These results strongly indicate that Glu-361 in human endothelial NOS is specifically involved in the interaction with L-arginine. Mutation of this residue abolished the L-arginine binding without disruption of other functional characteristics.Nitric oxide (NO) 1 has been identified as an important signal mediator that is involved in many physiological or pathophysiological processes. NO is produced together with L-citrulline through a two-step oxidation of L-arginine by three different NO synthase isoforms. The endothelial and neuronal isoforms are constitutively expressed, and their activities are regulated by calcium and calmodulin (Ca 2ϩ /CaM) (1, 2). The third NOS isoform is induced in response to cytokines or lipopolysaccharide, and its activity is independent of Ca 2ϩ /CaM (3). Despite the modest sequence homology and different regulation among the three NOS isoforms, they share a similar cofactor composition and possess a bidomain structure (4 -8). The C-terminal reductase domain is homologous to NADPH-cytochrome P450 reductase and has binding regions for NADPH, FAD, and FMN. The N-terminal oxygenase domain containing heme, BH 4 , and L-arginine binding sites is a P450-type hemoprotein, but does not show sequence homology to other known P450s. A CaM binding module exists near the center of the NOS sequences (9), and binding of Ca 2ϩ /CaM facilitates electron transfer from the reductase to the oxygenase domain (10, 11).Because of the obvious impact of NO on human health, a detailed understanding of the NOS reaction mechanism is essential for selective pharmacological intervention against individual isoforms. The oxygenase active site domain, including the proximal heme thiolate ligand, the distal heme pocket, and the substrate binding region is the center of NOS catalysis. Defining the amino acid residues making up the heme binding region and substrate oxidation site is a key s...

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“…These studies may suggest that endogenous ADP-ribosylation is under the regulatory control of second messenger systems, especially nitric oxide. Activation of nitric oxide synthase [24] in its various isoforms [25] produces nitric oxide, initially characterized as the endothelium-derived relaxing factor [26]. While some nitric oxide effects are mediated by binding of nitric oxide to the heme subunit of soluble guanylate cyclase, thus increasing cGMP [27], the stimulatory effects concerning the mono-ADPribosylation are cGMP independent and might be linked to higher and hence pathophysiological and cytotoxic nitric oxide conditions [28, 291. Our studies now further characterize this nitric-oxidecatalysed mono-ADP-ribosylation of GraPDH and explore some mechanistics of the ADP-ribose-transfer reaction.…”

mentioning

confidence: 99%

Characterization of a nitric‐oxide‐catalysed ADP‐ribosylation of glyceraldehyde‐3‐phosphate dehydrogenase

Dimmeler

Brüne

1992

European Journal of Biochemistry

128

102

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Auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GraPDH) has recently been demonstrated to be dramatically stimulated in the presence of nitric oxide. In order to obtain insight into the sequence of events leading to ADP-ribosylation of GraPDH, we studied the target amino acid, the nucleotide cofactor requirement, pH dependency and the stoichiometry of the reaction. Basal as well as stimulated ADP-ribose transfer is inhibited by the SH-group alkylating reagent, N-ethylmaleimide. Furthermore, the radiolabel of a~t o -[~~P ] A D pribosylated GraPDH is removed by treatment with HgC12, suggesting an ADP-ribose -cysteine bond. Several indirect and direct mechanistic considerations point to NAD' as the only cofactor for the ADP-ribosylation reaction, excluding the possibility of a reaction sequence involving a NADglycohydrolase(s) followed by nonenzymatic ADP-ribose transfer to GraPDH. Optimal ADPribosylations were carried out at alkaline pH values using 10 pM free NAD' as the sole nucleotide cofactor. Bovine serum albumin with an S-nitrosylated SH group can serve as a model of ADP-ribose transfer from NAD' and suggests that the nitric-oxide-modified SH group (S-nitrosylated SH group) is a prerequisite for the reaction. In a previous work, we demonstrated that nitric oxide was able to increase the mono-ADP-ribosylation of a 39-kDa cytosolic protein [18, 191, which was observed not only when nitric oxide was released from pharmacological compounds like sodium nitroprusside (SNP) or 3-morpholino-sydnonimine (SIN-l), but also when nitric oxide was generated from its physiological precursor L-arginine, by the enzymic activity of the nitric oxide synthase [22]. Nitric-oxide-stimulated ADP-ribosylation was recently confirmed by two other groups, using cerebral cortex and erythrocyte membranes [21, 231. These studies may suggest that endogenous ADP-ribosylation is under the regulatory control of second messenger systems, especially nitric oxide. Activation of nitric oxide synthase [24] in its various isoforms [25] produces nitric oxide, initially characterized as the endothelium-derived relaxing factor [26]. While some nitric oxide effects are mediated by binding of nitric oxide to the heme subunit of soluble guanylate cyclase, thus increasing cGMP [27], the stimulatory effects concerning the mono-ADPribosylation are cGMP independent and might be linked to higher and hence pathophysiological and cytotoxic nitric oxide conditions [28, 291. Our studies now further characterize this nitric-oxidecatalysed mono-ADP-ribosylation of GraPDH and explore some mechanistics of the ADP-ribose-transfer reaction. We would like to stress the importance of reduced thiol groups as a prerequisite for ADP-ribosylation, furthermore we would like to report on cysteine-specific modification, pH dependency and the specificity of NAD+, required for this ADPribosylation reaction.

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Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase

Cited by 2,257 publications

References 45 publications

Up‐regulation of nitric oxide synthase by estradiol in human aorticendothelial cells

Up‐regulation of nitric oxide synthase by estradiol in human aorticendothelial cells

Mutation of Glu-361 in Human Endothelial Nitric-oxide Synthase Selectively Abolishes L-Arginine Binding without Perturbing the Behavior of Heme and Other Redox Centers

Characterization of a nitric‐oxide‐catalysed ADP‐ribosylation of glyceraldehyde‐3‐phosphate dehydrogenase

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