Identification, characterization, and comparison of the calmodulin-binding domains of the endothelial and inducible nitric oxide synthases

Richard C. Venema, Hassan S. Sayegh, Jonathan D. Kent, David G. Harrison

Research output: Contribution to journalReview article

150 Scopus citations

Abstract

The calmodulin (CaM)-binding regions in bovine endothelial nitric oxide synthase (eNOS) and murine inducible nitric oxide synthase (iNOS) are identified in this study as eNOS residues 493-512 and iNOS residues 501-532. Peptides corresponding to eNOS 493-512 and iNOS 501-532 produce a Ca2+-dependent, electrophoretic mobility shift of CaM on 4 M urea gels. The two peptides are also potent inhibitors of the CaM-mediated activation of neuronal nitric oxide synthase and have dissociation constants for CaM binding of 4.0 and 1.5 nM, respectively. Substitution of eNOS and iNOS CaM-bind-ing domains in eNOS/iNOS chimeric proteins produces major alterations in the Ca2+ and CaM dependence of the intact enzymes expressed and purified from a baeuIovirus/Sf9 insect cell system. Replacement of aligned iNOS sequence with eNOS 493-512 creates a functional, chimeric iNOS that is both Ca2+- and CaM-dependent. Replacement of aligned eNOS sequence with iNOS 501-532 creates a functional, chimeric eNOS that is CaMindependent but that remains Ca2+-dependent. Specific amino acid residues critical for CaM binding by eNOS are also identified in this study as Phe-498, Lys-499, and Leu-511 in the bovine eNOS sequence.

Original languageEnglish (US)
Pages (from-to)6435-6440
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number11
DOIs
StatePublished - Mar 15 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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