Neuronal nitric oxide synthase isoforms alpha and mu are closely related calpain-sensitive proteins.
The neuronal nitric oxide synthase isoform nNOSmu, which is expressed in striated muscle, differs from nNOSalpha, the major brain isoform, by the insertion of 34 amino acid residues between the calmodulin- and flavin-binding domains [J Biol Chem 271:11204-11208 (1996)]. We show here that recombinant, purified nNOSmu, despite the peptide insertion, has the same spectroscopic properties, L-arginine kcat and Km values, optimal pH, and calmodulin binding affinity constant as nNOSalpha. However, nNOSmu consumes NADPH and reduces cytochrome c at approximately half the rate of nNOSalpha. The rates of degradation of the two proteins by rat brain and muscle homogenates show that nNOSmu is degraded more slowly than nNOSalpha. The in vitro half-lives of nNOSalpha and nNOSmu are 12 and 50 min, respectively, and calpain is important for this degradation. These short in vitro half-lives suggest that the nNOS isoforms are susceptible to rapid degradation in vivo. The elevated (20-fold) levels of calpain in diseased muscle tissue in Duchenne muscular dystrophy, and the hydrolytic sensitivity of both nNOS mu and nNOSalpha to this enzyme, may contribute to the deficiency of nNOS activity in the diseased tissue.
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446, USA.
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de Montellano
D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D002147:Calmodulin; D003574:Cytochrome c Group; D007527:Isoenzymes; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D002154:Calpain