Abstract
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.