The calmodulin-binding domain of the inducible (macrophage) nitric oxide synthase.
A domain in the inducible, macrophage nitric oxide (NO) synthase has been selected as the putative calmodulin-binding site. The domain was synthesized as a peptide of 29 residues [P29, NO synthase-(504-532)-peptide], having the accepted hydrophobic/basic composition of calmodulin-binding domains and containing, like most of them, an aromatic amino acid at its N-terminus and a long chain aliphatic residue 12 amino acids downstream of it. A 34-residue peptide from the synthase sequence [P34, NO synthase-(499-532)-peptide], consisting of peptide P29 and of the five extra N-terminal amino acids, three of them basic, was also synthesized. Both peptides bound calmodulin in the presence as well as in the absence of Ca2+ (i.e. in the presence of excess EGTA). The KD of the binding in the presence of Ca2+ was < or = 1 nM. The binding affinity was lower, but still remarkably high in the presence of EGTA. The peptides counteracted the stimulation by calmodulin of a classical calmodulin-target enzyme, the Ca2+ pump of the plasma membrane.
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
J
J
Anagli
F
F
Hofmann
M
M
Quadroni
T
T
Vorherr
E
E
Carafoli
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002148:Calmodulin-Binding Proteins / Q000138:chemical synthesis / Q000378:metabolism*; D004790:Enzyme Induction; D008264:Macrophages / Q000201:enzymology*; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000138:chemical synthesis / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment
D002148:Calmodulin-Binding Proteins; D019001:Nitric Oxide Synthase