Mutation of N-myristoylation site converts endothelial cell nitric oxide synthase from a membrane to a cytosolic protein.
Endothelial cell nitric oxide synthase (ECNOS) is a membrane-associated enzyme that generates endothelium-derived relaxing factor/nitric oxide (EDRF/NO) from L-arginine. We have suggested, from the cloning of the bovine ECNOS cDNA, that the presence of an N-myristoylation consensus sequence may impart its membrane localization since cytosolic forms of NOS do not contain such domains. To test the hypothesis that N-myristoylation is necessary for particulate ECNOS, we performed site-directed mutagenesis of the myristic acid acceptor site, Gly-2, and changed the glycine codon to alanine by a single nucleotide substitution. Expression of wild-type ECNOS in COS cells resulted in greater than 95% of the enzymatic activity in crude membrane fractions (as measured by the conversion of [3H]L-arginine to [3H]L-citrulline). In contrast, expression of the Gly-2 to Ala-2 mutant (G2A) demonstrated 8% ECNOS activity in membranes and 92% in the cytosol. The back mutation (from Ala-2 to Gly-2, A2G) restored ECNOS activity to the particulate fraction as seen with the wild type. Both wild-type membrane ECNOS and cytosolic G2A ECNOS activities were dependent on NADPH and calcium and were inhibited to the same extent by NG-monomethyl L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME). Moreover, kinetic analysis of these enzymes revealed similar Kms for L-arginine (2-4 microM, n = 3), demonstrating that the mutation did not affect ECNOS function. Thus, N-myristoylation is necessary for the membrane localization of ECNOS and may be of special significance for the basal or flow-induced production of NO by the endothelium.
Sessa
W C
WC
Department of Physiology, University of Virginia School of Medicine, Charlottesville.
Barber
C M
CM
Lynch
K R
KR
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000201:enzymology*; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D019001:Nitric Oxide Synthase; D014018:Tissue Distribution
D015335:Molecular Probes; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases