Ca2+/calmodulin-dependent cytochrome c reductase activity of brain nitric oxide synthase.
Nitric oxide acts as a widespread signal molecule and represents the endogenous activator of soluble guanylyl cyclase. In endothelial cells and brain tissue, NO is enzymatically formed from L-arginine by Ca2+/calmodulin-regulated NO synthases which require NADPH, tetrahydrobiopterin, and molecular oxygen as cofactors. Here we show that purified brain NO synthase binds to cytochrome c-agarose and exhibits superoxide dismutase-insensitive cytochrome c reductase activity with a Vmax of 10.2 mumol x mg-1 x min-1 and a Km of 34.1 microM. Cytochrome c reduction was largely dependent on Ca2+/calmodulin and cochromatographed with L-citrulline formation during gel filtration. When reconstituted with cytochrome P450, NO synthase induced a moderate Ca(2+)-independent hydroxylation of N-ethylmorphine. NO synthase also reduced the artificial electron acceptors nitro blue tetrazolium and 2,6-dichlorophenolindophenol. Cytochrome c, 2,6-dichlorophenolindophenol, and nitro blue tetrazolium inhibited NO synthase activity determined as formation of L-citrulline from 0.1 mM L-arginine in a concentration-dependent manner with half-maximal effects at 166, 41, and 7.3 microM, respectively. These results suggest that NO synthase may participate in cellular electron transfer processes and that a variety of electron-acceptors may interfere with NO formation due to the broad substrate specificity of the reductase domain of NO synthase.
Klatt
P
P
Institute of Pharmacology, Free University of Berlin, Germany.
Heinzel
B
B
John
M
M
Kastner
M
M
Böhme
E
E
Mayer
B
B
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002850:Chromatography, Gel; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010084:Oxidation-Reduction; D013482:Superoxide Dismutase / Q000378:metabolism; D013552:Swine
D002147:Calmodulin; D009580:Nitroblue Tetrazolium; D003577:Cytochrome P-450 Enzyme System; D015086:2,6-Dichloroindophenol; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; D000594:Amino Acid Oxidoreductases; D009245:NADH Dehydrogenase; D002118:Calcium