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eNOS activity assay

AH An He
SH Shupeng Hu
QP Qiangzhong Pi
YG Yongzheng Guo
YL Yang Long
SL Suxin Luo
YX Yong Xia
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eNOS activity was determined by converting rate of L-[14C] arginine to L-[14C] citrulline in cells. The conversion was monitored in a 300 μL buffer containing 50 mM Tris–HCl, pH 7.4, 5 μM L-[14C] arginine (Moravek, Brea, CA, USA), 45 μM L-arginine, 0.5 mM NADPH, 10 μM BH4, 10 μg/ml calmodulin, and 10 nM eNOS. The reactions were initiated by adding L-[14C] arginine and terminated in a stop buffer (20 mM HEPES, 2 mM EDTA) after incubation at 37 °C for 1 h. L-[14C] citrulline was separated from the reaction mixture by Dowex AG 50W-X8 (Na+ form) (Sigma) cation exchange columns and quantitated by liquid scintillation counting. N(gamma)-nitro-L-arginine methyl ester (L-NAME; 5 mM) inhibitory activity was analyzed to determine the concentration of L-[14C] citrulline converted by eNOS.

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