N-NITRO-L-ARGININE AND N-MONOMETHYL-L-ARGININE EXHIBIT A DIFFERENT PATTERN OF INACTIVATION TOWARD THE 3 NITRIC-OXIDE SYNTHASES

The ability of N-G-nitro-L-arginine (NNA) and N-G-methyl-L-arginine (NMMA) to inactivate native neuronal, endothelial cell, and macrophage nitric oxide synthases (nNOS, eNOS, and iNOS, respectively) was investigated. Each NOS isozyme (plus cofactors) was preincubated with either NNA or NMMA and then assayed for remaining activity by measuring the conversion of labeled L-arginine to labeled L-citrulline. Consistent with previous reports (Olken, N. M., et al, Biochem. Biophys. Res. Commun. 177, 828-833, 1991), NMMA was a mechanism-based irreversible inhibitor of iNOS, exhibiting time- and concentration-dependent inactivation of iNOS with a K-I equal to 2.6 mu M and a k(inact) equal to 0.042 min(-1). When assayed without a preincubation period, NMMA exhibited typical reversible inhibition of iNOS (K-i = 3.9 mu M). NMMA also reversibly inhibited nNOS and the eNOS with K-i equal to 0.65 and 0.7 mu M, respectively. However, NMMA did not inactivate eNOS at concentrations up to 10 mu M. In the presence, but not the absence, of 4 mu M tetrahydrobiopterin, NMMA inactivated nNOS with a k(inact) equal to 0.022 min(-1) and a K-I equal to 2.0 mu M. Since NNA did not inactivate iNOS at concentrations up to 25 mu M, NNA is strictly a reversible inhibitor of iNOS (K-i = 8.1 mu M). Neuronal NOS and eNOS, however, were rapidly inactivated by NNA with k(intact) equal to 0.083 and 0.047 min(-1) and K-I equal to 0.09 and 0.02 mu M, respectively, when preincubated with NNA. Tetrahydrobiopterin did not affect the rate of inactivation of nNOS by NNA. In all cases, L-arginine protected against inactivation, suggesting that inactivation occurs at or near the active site. Thus, inactivation of the three NOS isozymes with NMMA and MNA reveals active-site differences between the isoforms. (C) 1995 Academic Press, Inc.