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

Venema RC, Sayegh HS, Kent JD, Harrison DG
J Biol Chem. 1996 271 (11): 6435-40

PMID: 8626444 · DOI:10.1074/jbc.271.11.6435

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 NOS 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-binding domains in eNOS/iNOS chimeric proteins produces major alterations in the Ca2+ and CaM dependence of the intact enzymes expressed and purified from a baculovirus/Sf9 insect cell system. Replacement of aligned NOS sequence with eNOS 493-512 creates a functional, chimeric iNOS that is both (Ca2+)- and CaM-dependent. Replacement of aligned eNOS sequence with NOS 501-532 creates a functional, chimeric eNOS that is CaM-independent 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.

MeSH Terms (19)

Amino Acid Sequence Animals Baculoviridae Binding Sites Calcium Calmodulin Cattle Cell Line Chickens Endothelium Enzyme Induction Mice Molecular Sequence Data Nitric Oxide Synthase Rabbits Rats Recombinant Fusion Proteins Sequence Homology, Amino Acid Spodoptera

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