Identification of covalently bound amino-terminal myristic acid in endothelial nitric oxide synthase

Endothelial nitric oxide synthase (eNOS) is unique among the nitric oxide synthase family of proteins due to the presence of an N-myristoylation consensus sequence elucidated from the cloning of its cDNA. Although eNOS was metabolically labeled with [3H]myristic acid and mutation of glycine 2 in the N-myristoylation consensus sequence changed the particulate localization of the enzyme to a cytosolic form, the definitive characterization of eNOS as an N-myristoylprotein has not been demonstrated. Therefore, the purpose of the present study was to determine the nature of the fatty acid incorporated into eNOS. Wild-type or G2A mutant (mutation of glycine 2, the myristic acid acceptor site, to alanine) eNOS-transfected COS cells and bovine aortic endothelial cells (BAEC) were metabolically labeled with [3H]myristic acid for 5 h. The radiolabel was primarily incorporated into membrane-associated eNOS from wild-type transfected COS cells and cultured BAEC but not into the mutant eNOS from G2A-transfected COS cells. Qualitatively similar amounts of immunoreactive protein were found in wild-type and G2A-transfected cells. In addition, linkage of the radiolabel to eNOS was insensitive to hydroxylamine treatment, and incorporation of the radiolabel into eNOS was abolished by cyclo-heximide. Chemical analysis of the fatty acid released by acid methanolysis of labeled eNOS verified the 3H-labeled fatty acid as protein-bound myristic acid. These results unequivocally demonstrate that eNOS incorporates myristic acid via an amide linkage with the amino-terminal glycine of the enzyme as a co-translational modification.