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 oxide. In this study, wild-type or mutant form of rat nNOS was overexpressed in a baculovirus/Sf9 insect cell system and examined for autophosphorylation. Incubation of purified wild-type nNOS in the presence of Mg2+ and ATP result in phosphorylation of nNOS. Deletional mutant of the nNOS amino terminus which contains G-Q-G-A-G-S sequence, however, shows loss of phosphorylation. Furthermore, purified wild-type rat liver cytokine-induced NOS (iNOS), which does not contain G-Q-G-A-G-S sequence, 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 nNOS-specific autophosphorylation pathway might be involved in the regulation of enzyme activity.
Watanabe
Y
Y
Department of Pharmacology, Nagoya University School of Medicine, Showa-ku, Japan.
Terashima
K
K
Hidaka
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D019001:Nitric Oxide Synthase