Open AccessThe Calmodulin‐binding Domain of the Inducible (Macrophage) Nitric Oxide Synthase
Author(s) -
Anagli John,
Hofmann Francesco,
Quadroni Manfredo,
Vorherr Thomas,
Carafoli Ernesto
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.701_3.x
Subject(s) - calmodulin , egta , peptide , nitric oxide synthase , biochemistry , chemistry , peptide sequence , residue (chemistry) , enzyme , amino acid , binding site , calcium , organic chemistry , gene
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 Ca 2+ (i.e. in the presence of excess EGTA). The K D of the binding in the presence of Ca 2+ was ≤ 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 Ca 2+ pump of the plasma membrane.