Enzymatic formation of nitrogen oxides from L-arginine in bovine brain cytosol

Abstract

In dialyzed bovine brain cytosol, the enzymatic formation of nitrogen oxides was directly determined. The basal formation of nitrite and nitrate was concentration-dependently enhanced by L-arginine (EC50 about 3.10(-5) M). Both the basal and L-arginine induced formations were inhibited by NG-monomethyl-L-arginine (EC50 about 2.10(-4) M). In the presence of L-arginine, a concomitant formation of citrulline was detected. L-Arginine methyl ester also served as a substrate, but neither D-arginine, D-arginine methyl ester nor N alpha-benzoyl-L-arginine ethyl ester did so. The formation of nitrite and nitrate was time-dependent, increased linearly with the protein concentration of the cytosol and was not observed when the cytosolic proteins were heat-denaturated. Exogenous NADPH (or NADP+) concentration-dependently enhanced the formation of nitrite and nitrate, whereas NADH, NAD+, FAD, Ca2+, Mg2+ and calmodulin were ineffective. These results indicate that bovine brain contains a cytosolic enzyme which uses NADPH or NADP+ as cofactors to form nitrogen oxides from both an endogenous non-dialyzable substrate and from L-arginine.