N G-nitro-l-[ 3h]arginine binding properties of neuronal nitric oxide synthase in rat brain

Abstract

N G-Nitro-L-arginine (L-NNA), a derivative of L-arginine (L-Arg), is known as a pseudo-substrate and inhibitor for nitric oxide synthase (NOS). To clarify the regulatory mechanism of substrate-binding domain in neuronal NOS (nNOS), we examined the characteristics of N G-nitro-L-[ 3H]Arg (L-[ 3H]NNA) binding using the cytosolic fraction and purified nNOS from the rat cerebellum, in comparison with L-[ 14C]citrulline formation from L-[ 14C]Arg. The L-[ 3H]NNA binding was inhibited by L-NNA > N G-methyl-L-Arg > diphenyleneiodonium > L-Arg, but was not inhibited by L-citrulline and D-Arg. Thus, L-NNA seems to bind the substrate-binding domain in the nNOS with high affinity rather than L-Arg. Even in the absence of NADPH, tetrahydrobiopterin (BH 4) and Ca 2+, the L-[ 3H]NNA binding activity was observed in the cerebellar cytosol, although L-[ 14C]citrulline could not be produced from L-[ 14C]Arg. L-[ 3H]NNA binding was increased by BH 4 alone and was markedly enhanced by NADPH plus BH 4 (NADPH/BH 4), but not by Ca 2+/CaM. In contrast, L-[ 14C]citrulline was formed only in the presence of NADPH/BH 4 and Ca 2+. Similar results were obtained in purified nNOS. These results suggest that L-[ 3H]NNA seems to bind the substrate-binding domain in the nNOS but the binding affinity of L-Arg was lower than the affinity of L-NNA. Although the substrate binding is necessary to BH 4 and NADPH, Ca 2+/CaM are further necessary for the formation of NO and L-citrulline.