Ca2+-independent activity of nitric oxide synthase

Ca2+-independent forms of nitric-oxide synthase have significant activity w hen the endogenous calmodulin subunit is Ca2+ free, Further activation is s een when Ca2+ is added. We have examined the activation of a Ca2+-independe nt nitric-oxide synthase variant and its two point mutants that are more de pendent on Ca2+ for activation using mutant calmodulins containing non-func tional Ca2+-binding sites. These studies provide evidence that the Ca2+ ind ependent activity of these enzymes can be exerted through specific adapted interactions between the enzyme and the Ca2+-binding site 2 of calmodulin, Further, the results suggest that EGTA-sensitive metals other than Ca2+ com plexed to calmodulin may be involved in maximal activation of these nitric- oxide synthase variants. (C) 2001 Academic Press.