Interaction of calmodulin with the inducible murine macrophage nitric oxide synthase.
The inducible isoform of nitric oxide synthase (iNOS) isolated from murine macrophages copurifies with calmodulin (CaM) as a tightly bound subunit. The exact function of this tightly bound CaM, however, has not been elucidated. In order to probe the function of this unusually strong interaction between iNOS and CaM, a 30 amino acid peptide derived from the putative CaM binding site of iNOS was synthesized. Cross-linking and autoradiographic analyses demonstrated that the peptide and CaM form a 1:1 complex as well as several higher molecular weight complexes. When assayed in the presence of a 12-fold excess of peptide to iNOS, over 90% of the enzymatic activity was inhibited. This inhibition could be prevented with the addition of exogenous bovine CaM to the assay mixture, in a concentration-dependent manner. Native PAGE and Western blot analysis of iNOS treated with peptide revealed the formation of a peptide--CaM complex with CaM derived from iNOS. Moreover, EGTA (5 mM) caused a 30% maximal inhibition of activity that was reversed by the addition of exogenous Ca2+ in a concentration-dependent fashion, suggesting a role for Ca2+ in this interaction. EGTA also changed the native PAGE mobility of iNOS and increased the intensity of a band which comigrates with CaM. These results demonstrate that the binding interaction between CaM and iNOS is tight but reversible, requires Ca2+, and is atypical from other known CaM--enzyme interactions.
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology
D002147:Calmodulin; D010455:Peptides; D004533:Egtazic Acid; D019001:Nitric Oxide Synthase; D002118:Calcium