演題番号 : P1-b11
笠松 真吾 / Shingo Kasamatsu:1 渡邊 泰男 / Yasuo Watanabe:2 居原 秀 / Hideshi Ihara:1
1:大府大院・理・生物科学 / Dept of Biolo Sci, Grad Sch of Sci, Univ of Osaka Pref, Osaka 2:昭和薬科大学 薬理学部 / Dept of Pharmacol, Univ of Showa Pharm, Tokyo
[Background] Nitric oxide synthase (NOS) catalyzes the conversion of arginine (Arg) to citrulline (Cit) and NO, also produces reactive oxygen species (ROS) in the uncoupling reactions. It has been proved that neuronal NOS (nNOS) activities are reduced through the phosphorylation at the Ser847 of the enzyme. To date, no information about the effects of the phosphorylation is available. Here, in order to reveal the effects of the phosphorylation on the NO-ROS signaling, we prepared a Ser847D point mutant to mimic phosphorylation and examined the nNOS activities of the wild-type and the mutant in vitro and NO-ROS signaling in PC12 cells. [Method] We produced the mutant nNOS gene (S847D mutant). The wild-type nNOS and the mutant were overexpressed in E. coli, thereafter purified using 2',5'-ADP-Sepharose. We used a spectrophotometric assay to determine the NO synthesis and NADPH oxidation activities of the wild-type and the mutant forms. The uncoupling efficiency was calculated as the difference between the amount of NADPH consumed and the amount of Cit produced. The amount of Cit produced was determined using HPLC. We generated the PC12 cells expressed the nNOS and examined 8-nitro-cGMP generation. [Result] The NO synthesis activity was found to be 176 and 128 nmol mg-1min-1 in the wild-type and the mutant, respectively. The activity of the mutant was 27% lower than that of the wild-type. These results were consistent with the results of previous reports. The NADPH oxidation activity in the presence of Arg was 373 and 367 mg-1min-1 in the wild-type and the mutant, respectively. Further, in the absence of Arg, the activities were 643 and 846 mg-1min-1 in the wild-type and the mutant, respectively. The mutant activity was 32% higher than the wild-type activity. The uncoupling efficiency of the wild-type nNOS was 49% and that of the mutant was 62%. These results suggest that the phosphorylation at Ser847 of nNOS regulates the uncoupling efficiency.