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-mu-m), 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 K(m) values of 4.3-mu-m, 0.4-mu-M, 0.3-mu-M 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 N(G)-nitro-L-arginine, N(G)-methyl-L-arginine and Nitro Blue Tetrazolium with apparent K(i) values of 0.2, 1.8 and 7-mu-M 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. N(G)-Nitro-L-arginine and N(G)-methyl-L-arginine did not inhibit the cytochrome c reductase activity.