DISTINCT MOLECULAR RECOGNITION OF CALMODULIN-BINDING SITES IN THE NEURONAL AND MACROPHAGE NITRIC-OXIDE SYNTHASES - A SURFACE-PLASMON RESONANCE STUDY

The neuronal nitric oxide synthase and the macrophage nitric oxide syn thase are differently regulated by Ca2+/calmodulin. We investigated th e dynamics of calmodulin binding to the putative calmodulin-binding si tes In both nitric oxide synthases. Peptides derived from the putative calmodulin-binding sites were synthesized and immobilized to a dextra n layer of a biosensor chip. Complex formation of calmodulin and the p eptides was monitored by surface plasmon resonance spectroscopy and re corded as sensorgrams. We determined a dissociation constant K-D of 5. 0 x 10(-9) M for the neuronal nitric oxide synthase and calmodulin. Th e association rate constant and the dissociation rate constant were k( a) = 1.58 x 10(5) M(-1) s(-1) and k(d) = 7.87 x 10(-4) s(-1), respecti vely. Sensorgrams obtained with the macrophage nitric oxide synthase p eptide were remarkably different. Calmodulin, once bound to the peptid e, did not dissociate, Association of calmodulin to the peptide occurr ed with the same rate constants (k(a) = 3 x 10(4) M(-1) s(-1)) regardl ess of the presence or absence of Ca2+. The affinity was in the subnan omolar range (K-D < 0.1 x 10(-9) M). We conclude that the extremely ti ght binding of calmodulin to the NOS-II is solely controlled by the ca lmodulin-binding segment and not by other parts of tile protein.