Identification and characterization of a calmodulin-dependent nitric oxide synthase from GH3 pituitary cells.
A nitric oxide synthase activity stimulated more than 30-fold by the concurrent presence of Ca2+ and calmodulin (CaM), and inhibited by trifluoperazine (50 microM), has been identified in extracts of GH3 pituitary cells. The CaM-dependent nitric oxide synthase of the crude extract was stimulated more than 9-fold by (6R)-5,6,7,8-tetrahydro-L-biopterin with half-maximal stimulation occurring at a concentration of 300 nM. Fractionation of the extract on DEAE-cellulose enhanced nitric oxide synthase specific activity up to 300-fold and provided a preparation which on Western blot analysis possessed a 152 kDa protein which cross-reacted with antibodies to homogeneous bovine brain nitric oxide synthase. The DEAE-cellulose-purified enzyme exhibited apparent Km values of 4.3 microM, 0.4 microM, 0.3 microM and 4 nM for L-arginine, NADPH, Ca2+ and CaM respectively. The CaM-dependent nitric oxide synthase of GH3 extract bound to 2',5'-ADP-agarose and was eluted by NADPH with a 500-fold increased specific activity. Citrulline formation by the ADP-agarose-purified enzyme was inhibited by NG-nitro-L-arginine, NG-methyl-L-arginine and Nitro Blue Tetrazolium with apparent Ki values of 0.2, 1.8 and 7 microM respectively. The ADP-agarose-purified enzyme displayed cytochrome c reductase activity which was stimulated more than 18-fold by the concurrent presence of Ca2+ and CaM and inhibited by trifluoperazine. NG-Nitro-L-arginine and NG-methyl-L-arginine did not inhibit the cytochrome c reductase activity.
Wolff
D J
DJ
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854.
Datto
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GA
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001345:Autoradiography; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002848:Chromatography, DEAE-Cellulose; D002956:Citrulline / Q000096:biosynthesis; D004789:Enzyme Activation; D007700:Kinetics; D009245:NADH Dehydrogenase / Q000378:metabolism; D019001:Nitric Oxide Synthase; D019335:Nitroarginine; D009580:Nitroblue Tetrazolium / Q000494:pharmacology; D010902:Pituitary Gland / Q000166:cytology / Q000201:enzymology*; D019323:omega-N-Methylarginine
D002147:Calmodulin; D019335:Nitroarginine; D001708:Biopterins; D019323:omega-N-Methylarginine; D009580:Nitroblue Tetrazolium; D002956:Citrulline; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D009245:NADH Dehydrogenase; C003402:sapropterin