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.
Liu
J
J
Molecular Cardiobiology Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812.
Sessa
W C
WC
D000409:Alanine; D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology*; D005998:Glycine; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000032:analysis / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000032:analysis / Q000378:metabolism; D017354:Point Mutation; D017368:Protein Prenylation; D014162:Transfection; D014316:Tritium
D009227:Myristic Acids; D010169:Palmitic Acids; D019814:Myristic Acid; D014316:Tritium; D019308:Palmitic Acid; D003513:Cycloheximide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D000409:Alanine; D005998:Glycine