NEURONAL NITRIC-OXIDE SYNTHASE SPECIFIC AUTOPHOSPHORYLATION IN BACULOVIRUS SF9 INSECT-CELL SYSTEM/

Neuronal Nitric Oxide Synthase (nNOS) is a calmodulin (CaM)-dependent enzyme, which generates the ubiquitous biological mediator nitric oxid e. In this study, wild-type or mutant form of rat nNOS was overexpress ed in a baculovirus/Sf9 insect cell system and examined for autophosph orylation. Incubation of purified wild-type nNOS in the presence of Mg 2+ and ATP result in phosphorylation of nNOS. Deletional mutant of the nNOS amino terminus which contains G-Q-G-A-G-S sequence, however, sho ws loss of phosphorylation. Furthermore, purified wild-type rat liver cytokine-induced NOS (iNOS), which does not contain G-Q-G-A-G-S sequen ce, shows no phosphorylation activity. We also found that the ratio of wild-type nNOS phosphorylation was independent of nNOS concentration, consistent with an intramolecular reaction. These data suggest that n NOS-specific autophosphorylation pathway might be involved in the regu lation of enzyme activity. (C) 1996 Academic Press, inc.