title: Distinct molecular recognition of calmodulin-binding sites in the neuronal and macrophage nitric oxide synthases: a surface plasmon resonance study.
abstract: The neuronal nitric oxide synthase and the macrophage nitric oxide synthase are differently regulated by Ca2+/calmodulin. We investigated the dynamics of calmodulin binding to the putative calmodulin-binding sites in both nitric oxide synthases. Peptides derived from the putative calmodulin-binding sites were synthesized and immobilized to a dextran layer of a biosensor chip. Complex formation of calmodulin and the peptides was monitored by surface plasmon resonance spectroscopy and recorded as sensorgrams. We determined a dissociation constant KD of 5.0 x 10(-9) M for the neuronal nitric oxide synthase and calmodulin. The association rate constant and the dissociation rate constant were ka = 1.58 x 10(5) M-1 s-1 and kd = 7.87 x 10(-4) s-1, respectively. Sensorgrams obtained with the macrophage nitric oxide synthase peptide were remarkably different. Calmodulin, once bound to the peptide, did not dissociate. Association of calmodulin to the peptide occurred with the same rate constants (ka = 3 x 10(4) M-1 s-1) regardless of the presence or absence of Ca2+. The affinity was in the subnanomolar range (KD) < 0.1 x 10(-9) M). We conclude that the extremely tight binding of calmodulin to the NOS-II is solely controlled by the calmodulin-binding segment and not by other parts of the protein.
authors.affiliation: Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Zoche
authors.forename: M
authors.initials: M
authors.lastname: Bienert
authors.forename: M
authors.initials: M
authors.lastname: Beyermann
authors.forename: K W
authors.initials: KW
authors.lastname: Koch
mesh_terms: D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013057:Spectrum Analysis
chemical_list: D002147:Calmodulin; D019001:Nitric Oxide Synthase