Characterization of Neuronal Nitric Oxide Synthase and a C415H Mutant, Purified from a Baculovirus Overexpression System

Richards, Michael; Marletta, Michael A.

doi:10.1021/bi00253a010

Cited by 101 publications

(93 citation statements)

References 44 publications

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“…After incubation at 37°C for 3 min, the conversion of arginine to citrulline was determined by ion exchange purification as described previously (18). The K m determined for arginine by this method was 4.4 M, which is consistent with that found by other laboratories (21,22).…”

Section: Methodssupporting

confidence: 92%

Guanabenz-mediated Inactivation and Enhanced Proteolytic Degradation of Neuronal Nitric-oxide Synthase

Noguchi¹,

Jianmongkol

Bender³

et al. 2000

Journal of Biological Chemistry

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Nitric-oxide synthases (NOS)1 are cytochrome P450-like hemoprotein enzymes that catalyze the conversion of L-arginine to citrulline and nitric oxide (1-4). Nitric oxide is a signaling molecule that is involved in a variety of physiological processes, including neurotransmission, vasorelaxation, platelet aggregation, and penile erection as well as in a variety of pathological conditions including septic shock, reperfusion injury, arthritis, atherosclerosis, diabetes, and graft rejection (5-8). It has been noted (9) that because nitric oxide is not stored, released, or inactivated after synaptic release by conventional regulatory mechanisms the biosynthetic regulation of the enzyme is of great importance. For the neuronal isoform the Ca 2ϩ -mediated activation is of prime importance. However, the factors that regulate proteolytic degradation of the enzyme have not been investigated. One approach that has been successfully utilized for the study of the proteolytic turnover of other P450 cytochromes is the use of suicide inactivators (10 -13). We wished to utilize this approach for the study of NOS turnover.Suicide inactivators are chemically inert molecules that mimic the natural substrate of the enzyme and become metabolized to a highly reactive intermediate that can covalently alter important active site entities, resulting in inactivation of the enzyme (14). In effect, the compound causes the enzyme to catalyze its own demise. Because the compound must not only have affinity for the active site but also must be metabolized in a manner to generate a reactive intermediate that is in close proximity to an important active site entity, these agents have the potential to be highly specific in vivo. In addition, they react with the form of the enzyme that is engaged in catalysis and thus are useful mechanistic probes into the nature of the bioactivation reaction. In some cases, these agents covalently alter P450 cytochromes in a manner that enhances their proteolysis and turnover (10 -13, 15-17).Guanabenz, a clinically used antihypertensive agent with a guanidino moiety, was recently shown, with the use of brain and penile cytosol, to be a metabolism-based inactivator of neuronal nitric-oxide synthase (nNOS) (18). Moreover, the treatment of rats with guanabenz was found to cause not only a decrease in activity, but also a concomitant loss of immunodetectable nNOS protein. To further understand the molecular mechanisms responsible for the loss of nNOS protein in vivo, we chose to model the effects of guanabenz with the use of HEK 293 cells stably transfected with nNOS. In the current study, we have shown that guanabenz causes the enhanced proteolytic turnover of nNOS. Suicide inactivators of nNOS, such as N G -methyl-L-arginine or N 5 -(1-iminoethyl)-L-ornithine, also caused the enhanced proteolytic degradation of the enzyme, whereas the slowly reversible inhibitor N G -nitro-L-arginine or the reversible inhibitor 7-NI did not. Studies with protease inhibitors indicate that the proteasome is responsible, in part, for r...

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Section: Methodssupporting

confidence: 92%

Guanabenz-mediated Inactivation and Enhanced Proteolytic Degradation of Neuronal Nitric-oxide Synthase

Noguchi¹,

Jianmongkol

Bender³

et al. 2000

Journal of Biological Chemistry

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“…This region contains the presumptive heme-and calmodulinbinding sites in mammalian NOS enzymes. The heme-binding site was proposed to be centered around C-415 in the nNOS (35,36), and this cysteine residue has been shown to be important for heme binding (37). The sequence surrounding the corresponding cysteine (C-329) in DNOS is well conserved.…”

Section: Resultsmentioning

confidence: 99%

Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase.

Regulski

Tully²

1995

Proc. Natl. Acad. Sci. U.S.A.

227

146

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Nitric oxide (NO) is an intercellular messenger involved with various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). Here, we report the cloning of a Drosophila NOS gene, dNOS, located at cytological position 32B.The dNOS cDNA encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, DNOS protein contains putative binding sites for calmodulin, FMN, FAD, and NADPH. DNOS activity is Ca2+/calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates.Nitric oxide (NO) is synthesized by NO synthases (NOSs) during conversion of L-arginine to L-citrulline (for reviews, see refs. 1 and 2). Biochemical characterization of NOSs has distinguished two general classes: (i) constitutive, dependent on exogenous Ca2+/calmodulin, and (ii) inducible, independent of exogenous Ca2+/calmodulin. Analyses of cDNA clones have identified three distinct NOS genes in mammals (3-6): neuronal, endothelial, and macrophage. The neuronal and endothelial NOSs are constitutive, and the macrophage NOS is inducible. The nomenclature for these different isoforms used here is historical, as it is clear now that one or more isoforms can be present in the same tissue (7).As a diffusible, free-radical gas, NO is a multifunctional messenger affecting many diverse aspects of mammalian physiology (for review, see ref. 8), such as regulation of vascular tone, macrophage-mediated cytotoxicity, and cell-cell interactions in the nervous system, including synaptogenesis and apoptosis during development of the rat central nervous system (9) and during neuronal cell differentiation (10). NO also appears to be involved with long-term potentiation in hippocampus and with long-term depression in cerebellum, two forms of synaptic plasticity that may underlie behavioral plasticity (11-15). Consistent with these cellular studies, inhibition of NOS activity may disrupt learning (refs. 16-19, but see refs. 20 and 21).Many of the above results are based on pharmacological studies using inhibitors of NOS or donors of NO. Interpretations of such studies usually are limited because the drugs interact with more than one target and cannot be delivered to specific sites. A molecular genetic approach can overcome these problems, however, by disrupting a specific gene, the product of which may be one of the drug's targets. Recently, such an approach has been attempted in mice via generation of a knockout mutation of the neuronal NOS (nNOS) (22).While nNOS mutants appeared fully viable and fertile, minor defects in stomach morphology and hippocampal long-term potentiation were detected (22, 23). Taken together, the above reports suggest roles for NO in developmental and behav...

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“…Defining the amino acid residues making up the heme binding region and substrate oxidation site is a key step in unraveling the biochemistry of NO synthesis. The proximal heme thiolate ligand was first predicted by McMillan et al (12) and subsequently confirmed by site-directed mutagenesis and spectral analysis for three NOS isoforms (13)(14)(15)(16). However, due to the lack of a three-dimensional NOS structure and the lack of sequence homology of the NOS oxygenase domain to other P450s, it has been difficult to identify residues in the distal heme pocket responsible for L-arginine binding.…”

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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Characterization of Neuronal Nitric Oxide Synthase and a C415H Mutant, Purified from a Baculovirus Overexpression System

Cited by 101 publications

References 44 publications

Guanabenz-mediated Inactivation and Enhanced Proteolytic Degradation of Neuronal Nitric-oxide Synthase

Guanabenz-mediated Inactivation and Enhanced Proteolytic Degradation of Neuronal Nitric-oxide Synthase

Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase.

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

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