PIN inhibits nitric oxide and superoxide production from purified neuronal nitric oxide synthase

A protein inhibitor of neuronal nitric oxide synthase (nNOS) was identified and designated as PIN. PIN was reported to inhibit nNOS activity in cell lysates through disruption of enzyme dimerization. However, there has been lack of direct characterization of the effect of PIN on NO production from purified nNOS. Furthermore, nNOS also generates superoxide (O2−) at low levels of l-arginine. It is unknown whether PIN affects O2− generation from nNOS. Therefore, we performed direct measurements of the effects of PIN on NO and O2− generation from purified nNOS using electron paramagnetic resonance spin trapping techniques. nNOS was isolated by affinity chromatography and a fusion protein CBP-PIN was used to probe the effect of PIN. While the tag CBP did not affect nNOS activity, CBP-PIN caused a dose-dependent inhibition on both NO and l-citrulline production. In the absence of l-arginine, strong O2− generation was observed from nNOS, and this was blocked by CBP-PIN in a dose-dependent manner. With low-temperature polyacrylamide gel electrophoresis, neither CBP nor CBP-PIN was found to affect nNOS dimerization. Thus, these results suggested that PIN not only inhibits NO but also O2− production from nNOS, and this is through a mechanism other than decomposition of nNOS dimers.