Superoxide Anion in the Enzymatic Conversion of L-Arginine to NO

Involvement of Superoxide Anion in the Enzymatic Conversion of L-Arginine to NO 

The original clue to the endogenous formation of NO in a biological system came—but was universally overlooked at the time—from the studies 

On the other hand, the iNOS isoform, which is associated with an adaptation phenomenon, has been reported to be calcium independent (Forstermann et al., 1991b). 

Reports dealing with mechanistic studies of NOS indicate that the enzyme can transfer electrons from NADPH to molecular oxygen via FAD or FMN to form superoxide anion and H2O2 in the presence of calcium or calmodulin (Mayer et al., 1990 Pou etal., 1992). One of these studies also demonstrated the concentration-dependent diminution of superoxide anion generation in the presence of N -nitro-L-arginine methyl ester (l-NAME). NOS has also been shown to donate electrons to acceptors, including nitro-blue tetrazolium (NBT) (Dawson et al., 1991) and cytochrome c (Klatt et al., 1992). These studies have used L-citrulhne rather than NO determination to detect NOS activity. 

The addition of NBT, which is known to interact with the superoxide anion and is reduced to formazan, led to the concentration-dependent inhibition of NOS activity, with an apparent K of 3-4 p.M (Mittal et al., 1993). The inhibitory effect of NBT was compared with N -monomethyl-L-arginine (NMA), a structural analog of L-arginine. Inclusion of NMA or NBT at 100 fjiM led to the complete inhibition of L-arginine-dependent stimulation of guanylate cyclase activity without any effect on the basal cGMP produc- 

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