Endothelial nitric oxide synthase. N-terminal myristoylation determines subcellular localization.

Nitric oxide synthases in diverse mammalian tissues catalyze the oxidation of L-arginine to L-citrulline plus nitric oxide (NO). In the vascular endothelium, synthesis of NO yields a labile intercellular messenger molecule with potent biological activities, including vascular smooth muscle relaxation. We have recently documented that the endothelial cell NO synthase (EC-NOS) constitutes a genetically distinct tissue-specific enzyme isoform. In further contrast to the soluble NO synthases found in neural tissues and in macrophages, the endothelial enzyme is associated primarily with the particulate fraction. Analysis of molecular clones for the endothelial NO synthase reveals no obvious transmembrane-spanning region, but a consensus motif for N-terminal myristoylation was identified; such a consensus sequence is not evident in the primary sequence of the soluble macrophage and neural NO synthases. We performed oligonucleotide-directed mutagenesis of the myristoylation consensus sequence in the endothelial NO synthase cDNA, and studied the pattern of expression of the wild-type and mutant EC-NOS cDNAs in transient transfection experiments in COS-7 cells. The subcellular localization of heterologous endothelial NO synthase was determined using analyses of enzyme activity as well as immunoprecipitation of biosynthetically labeled NO synthase with a highly specific antipeptide antibody. Expression of the wild-type endothelial NO synthase cDNA in COS-7 cells results in targeting of both enzyme activity and NO synthase immunoreactivity primarily to the particular fraction. By contrast, transient expression of the myristoylation- mutant cDNA in COS-7 cells yields NO synthase enzyme activity and immunoreactivity associated exclusively with the cytosol fraction. Following biosynthetic labeling with [3H]myristate, the NO synthase can be specifically immunoprecipitated from the particulate fraction in endothelial and in COS-7 cells transfected with the wild-type cDNA, but not in cells transfected with the myristoylation- mutant EC-NOS cDNA. N-terminal myristoylation of the endothelial NO synthase may provide a potential point of regulation of the biological functions of endothelium-derived NO in situ.