Calmodulin promotes dimerization of the oxygenase domain of human endothelial nitric-oxide synthase.
The active form of endothelial nitric-oxide synthase (eNOS) is a homodimer. The activity of the enzyme is regulated in vivo by calcium signaling involving the binding of calmodulin (CAM), which triggers the activation of eNOS. We have examined the possible role of calcium-mediated CAM binding in promoting dimerization of eNOS through the oxygenase domain of the enzyme. A recombinant form of the oxygenase domain of human eNOS was expressed in a prokaryotic expression system. This recombinant domain contains the catalytic cytochrome P-450 site for arginine oxidation by molecular oxygen as well as the binding sites for tetrahydrobiopterin and Ca2+-CAM but lacks the reductase domain and associated FAD, FMN, and NADPH binding sites. Binding of Ca2+-CAM caused an association of monomeric eNOS oxygenase domain as determined by changes in fluorescence, both intrinsic and extrinsic, and by gel filtration, chemical cross-linking, and particle-sizing. Dimerization of the domain was not dependent on the presence of the substrate, arginine, or the cofactor, tetrahydrobiopterin. A truncated form of the eNOS oxygenase domain lacking the Ca2+-CAM binding region did not undergo self-association to form dimers. These results show that the eNOS reductase domain is not required for Ca2+-CAM-induced dimerization of eNOS and suggest that this dimerization may be a primary event in the activation of eNOS by Ca2+.
Hellermann
G R
GR
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, Florida 33612, USA.
Solomonson
L P
LP
D002147:Calmodulin / Q000494:pharmacology*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D019281:Dimerization; D004730:Endothelium, Vascular / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010105:Oxygenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D011994:Recombinant Proteins; D010105:Oxygenases; D019001:Nitric Oxide Synthase