tailieunhanh - báo cáo khoa hoc : Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin

Using interferometry-based biosensors the binding and release of endothe-lial and neuronal nitric oxide synthase (eNOS and nNOS) from calmodulin (CaM) was measured. In both isoforms, binding to CaM is diffusion lim-ited and within approximately three orders of magnitude of the Smolu-chowski limit imposed by orientation-independent collisions. | IFEBS Journal Rate affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin Jonathan L. McMurry1 Carol A. Chrestensen1 Israel M. Scott1 Elijah W. Lee2 Aaron M. Rahn2 Allan M. Johansen1 Brian J. Forsberg1 Kyle D. Harris1 and John C. Salerno1 2 1 Department of Chemistry Biochemistry Kennesaw State University Kennesaw GA USA 2 Department of Biology Kennesaw State University Kennesaw GA USA Keywords calmodulin kinase nitric oxide synthase optical biosensing protein kinase C Correspondence J. C. Salerno Department of Biology MB 1202 1000 Chastain Rd Kennesaw GA 30144 USA Fax 1 770 423 6625 Tel 1 770 423 6177 E-mail jsalern3@ Received 1 April 2011 revised 8 August 2011 accepted 16 September 2011 doi Using interferometry-based biosensors the binding and release of endothelial and neuronal nitric oxide synthase eNOS and nNOS from calmodulin CaM was measured. In both isoforms binding to CaM is diffusion limited and within approximately three orders of magnitude of the Smolu-chowski limit imposed by orientation-independent collisions. This suggests that the orientation of CaM is facilitated by the charge arrays on the CaM-binding site and the complementary surface on CaM. Protein kinase C phosphorylation of eNOS T495 adjacent to the CaM-binding site abolishes or greatly slows CaM binding. Kinases which increase the activity of eNOS did not stimulate the binding of CaM which is already diffusion limited. The coupling of Ca2 binding and CaM NOS binding equilibria links the affinity of CaM for NOS to the Ca2 dependence of CaM binding. Hence changes in the Ca2 sensitivity of CaM binding always imply changes in the NOS-CaM affinity. It is possible however that in some regimes binding and activation are not synonymous so that Ca2 sensitivity need not be tightly linked to CaM sensitivity of activation. This study is being extended using mutants to probe the roles of .