Lainé R, de Montellano P R
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446, USA.
Mol Pharmacol. 1998 Aug;54(2):305-12. doi: 10.1124/mol.54.2.305.
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.
在横纹肌中表达的神经元型一氧化氮合酶亚型nNOSmu，与主要的脑型亚型nNOSalpha不同，在钙调蛋白结合域和黄素结合域之间插入了34个氨基酸残基[《生物化学杂志》271:11204 - 11208(1996)]。我们在此表明，重组纯化的nNOSmu尽管有肽段插入，但具有与nNOSalpha相同的光谱性质、L - 精氨酸催化常数和米氏常数、最佳pH值以及钙调蛋白结合亲和常数。然而，nNOSmu消耗NADPH并还原细胞色素c的速率约为nNOSalpha的一半。大鼠脑和肌肉匀浆对这两种蛋白质的降解速率表明，nNOSmu的降解比nNOSalpha慢。nNOSalpha和nNOSmu在体外的半衰期分别为12分钟和50分钟，钙蛋白酶对此降解过程很重要。这些较短的体外半衰期表明nNOS亚型在体内易快速降解。杜兴氏肌营养不良症患病肌肉组织中钙蛋白酶水平升高（20倍），以及nNOSmu和nNOSalpha对该酶的水解敏感性，可能导致患病组织中nNOS活性缺乏。
