N-G-Nitro-L-[H-3]arginine binding properties of neuronal nitric oxide synthase in rat brain

N-G-Nitro-L-arginine (L-NNA), a derivative of L-arginine (L-Arg), is known as a pseudosubstrate 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-[H-3]Arg (L-[H-3]NNA) binding using the cytosolic fraction and purified nNOS from the rat cerebellum, in comparison with L-[C-14]citrulline formation From L-[C-14]Arg. The L-[H-3]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 (BH4) and Ca2+, the L-[H-3]NNA binding activity was observed in the cerebellar cytosol, although L-[C-14]citrulline could not be produced from L-[C-14]Arg. L-[H-3]NNA binding was increased by BH4 alone and was markedly enhanced by NADPH plus BH4 (NADPH/BH4), but not by Ca2+/CaM. In contrast, L-[C-14]citrulline was formed only in the presence of NADPH/BH4 and Ca2+. 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 BH4 and NADPH, Ca2+/CaM are further necessary for the formation of NO and L-citrulline. (C) 1997 Elsevier Science Ltd. All rights reserved.