DIRECT MEASUREMENT OF NITRIC-OXIDE GENERATION FROM NITRIC-OXIDE SYNTHASE

Although nitric oxide synthase (NOS) is widely considered as the major source of NO in biological cells and tissues, direct evidence demonst rating NO formation from the purified enzyme has been lacking. It was recently reported that NOS does not synthesize NO, but rather generate s nitroxyl anion (NO-) that is subsequently converted to NO by superox ide dismutase (SOD), To determine if NOS synthesizes NO, electron para magnetic resonance (EPR) spectroscopy was applied to directly measure NO formation from purified neuronal NOS. In the presence of the NO tra p Fe2+-N-methyl-D-glucamine dithiocarbamate, NO gives rise to characte ristic EPR signals with g = 2.04 and a(N) = 12.7 G, whereas NO-is unde tectable. In the presence of L-arginine (L-Arg) and cofactors, NOS gen erated prominent NO signals, This NO generation did not require SOD, a nd it was blocked by the specific NOS inhibitor N-nitro-L-arginine met hyl ester, Isotope-labeling experiments with L-[N-15]Arg further demon strated that NOS-catalyzed NO arose from the guanidino nitrogen of L-A rg, Measurement of the time course of NO formation demonstrated that i t paralleled that of L-citrulline. The conditions used in the prior st udy were shown to result in potent superoxide generation, and this may explain the failure to measure NO formation in the absence of SOD. Th ese experiments provide unequivocal evidence that NOS does directly sy nthesize NO from L-Arg.