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

Yong Xia, Carlos O. Berlowitz, Jay L. Zweier

Research output: Contribution to journalArticlepeer-review

Abstract

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 ({radical dot}O2-) at low levels of l-arginine. It is unknown whether PIN affects {radical dot}O2- generation from nNOS. Therefore, we performed direct measurements of the effects of PIN on NO and {radical dot}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 {radical dot}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 {radical dot}O2- production from nNOS, and this is through a mechanism other than decomposition of nNOS dimers.

Original languageEnglish (US)
Pages (from-to)1445-1449
Number of pages5
JournalBiochimica et Biophysica Acta - General Subjects
Volume1760
Issue number9
DOIs
StatePublished - Sep 2006
Externally publishedYes

Keywords

  • Dimerization
  • Nitric oxide
  • Nitric oxide synthase
  • PIN
  • Superoxide

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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