Identification, characterization, and comparison of the calmodulin-binding domains of the endothelial and inducible nitric oxide synthases.
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.
Venema
R C
RC
Vascular Biology Center, Medical College of Georgia, Augusta 30912, USA.
Sayegh
H S
HS
Kent
J D
JD
Harrison
D G
DG
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002645:Chickens; D004727:Endothelium / Q000201:enzymology; D004790:Enzyme Induction; D051379:Mice; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D019001:Nitric Oxide Synthase; D002118:Calcium