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.
In the presence of its allosteric activator GDP, the major phosphofructokinase-1 from Escheric/ziu co/i K12 follows Michaelis-Menten kinetics.The kinetic behavior observed at steady-state using different concentrations of the substrates ATP and fructosed-phosphate and the pattern of inhibition by the substrate analogs adenylyl-@,y-methylene)-diphosphonate and D-arabinose-5-phosphate are consistent with a random sequential mechanism in rapid equilibrium, rather than with an ordered binding as was suggested earlier. However, ATP and fructose-6-phosphate do not bind independently to the same active site, since the apparent affinity for one substrate is decreased about 20-fold when the other substrate is already bound. The antagonism between ATP and fructose-6-phosphate shows that a negative interaction occurs during the reaction with E. co/i phosphofructokinase-1 which must be considered in addition to its allosteric properties.
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