NG-nitro-L-[3H]arginine binding properties of neuronal nitric oxide synthase in rat brain.
NG-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 NG-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 > NG-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-[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 BH4 alone and was markedly enhanced by NADPH plus BH4 (NADPH/BH4), but not by Ca2+/CaM. In contrast, L-[14C]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.
Arima
T
T
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Kitamura
Y
Y
Nishiya
T
T
Kiriyama
Y
Y
Taniguchi
T
T
Nomura
Y
Y
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009474:Neurons / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019335:Nitroarginine / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D002147:Calmodulin; D019335:Nitroarginine; D002956:Citrulline; D009249:NADP; D019001:Nitric Oxide Synthase; D002118:Calcium