Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase.
Endothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO), is derived from a guanidino nitrogen of L-arginine via its metabolism by nitric oxide synthase (NOS). Herein, we report the molecular cloning of a cDNA encoding the constitutive calcium-calmodulin (Ca2+/CaM)-regulated nitric oxide synthase (ECNOS). A full-length ECNOS clone was isolated by screening a bovine aortic endothelial cell cDNA library using a fragment of rat brain NOS (bNOS) cDNA. This cDNA has an open reading frame of 3615 nucleotides encoding a 1205-amino acid protein. Membranes prepared from COS cells transfected with the ECNOS cDNA demonstrated NADPH- and Ca2+/CaM- dependent conversion of L-, but not D-, arginine to NO and citrulline that was inhibited by NG-nitro-L-arginine methyl ester. Comparison of the deduced amino acid sequence of ECNOS to the bNOS and macrophage NOS (Mac-NOS) sequences revealed 57 and 50% identity, respectively. In addition, ECNOS contains a unique N-myristylation consensus sequence (not shared by bNOS or Mac-NOS) that may explain its membrane localization.
Sessa
W C
WC
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908.
Harrison
J K
JK
Barber
C M
CM
Zeng
D
D
Durieux
M E
ME
D'Angelo
D D
DD
Lynch
K R
KR
Peach
M J
MJ
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D002531:Cerebellum / Q000201:enzymology; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004730:Endothelium, Vascular / Q000201:enzymology*; D015870:Gene Expression; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D019001:Nitric Oxide Synthase; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D009838:Oligodeoxyribonucleotides; D012313:RNA; D004247:DNA; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases