Characterization of EGTA-washed synaptosomal membrane with emphasis on its calmodulin-binding proteins. Demonstration of possible reconstitution with added calcium/calmodulin

Abstract

Endogenous calmodulin (CaM) in the EGTA-washed cerebral-cortical synaptosomal membrane (SM) preparation was estimated below 3 μg/ml protein by the semiquantitative immunoblot analysis (Natsukari, N., Ohta, H. and Fujita, M. (1989) J. Immunol. Methods 125, 159–166). Membrane-bound CaM was immunoelectron-microscopically demonstrated in EGTA-washed, non-treated (control), and Ca 2+-treated cerebral-cortical synaptosomal membranes (SM) as well as for the SM enriched with added CaM. The density of CaM increased in the above order. CaM-dependent adenylate cyclase and CaM-dependent protein kinase II (CaM-kinase II) activities were restored, whereas the phosphodiesterase (PDE) activity was not affected by exogenous CaM over all the Ca 2+ concentrations tested. Adenylate cyclase at p Ca 6.2 was synergistically activated either by GTP and CaM or by CaM and β-adrenergic agonist, (±)-isoproterenol, reflecting the intactness of signal transduction pathway in the SM. Also demonstrated were the presence of protein kinase A, CaM-kinase II, and their endogenous substrates in the SM. Based on 32P-autoradiography and 125I-CaM overlay data certain CaM-binding proteins such as CaM-kinase II and synapsin I were identified on SDS-PAGE. Ca 2+-dependent and -independent CaMBPs were distinguished by 125I-CaM gel overlay with and without Ca 2+. The former had bigger molecular size (≧ 49 kDa) than the latter (≦ 34 kDa). Yield of Ca 2+-dependent CaMBPs was not affected by Ca 2+ concentration during preparation of the SM while that of Ca 2+-independent CaMBI's was reduced by exposure to 100 μM Ca 2+. In contrast with the CaMBPs of brain SM, those of enterocyte and erythrocyte plasma membranes especially, microvillous membrane of the enterocyte, showed quite distinct CaMBP profiles. The present findings suggested that the EGTA-washed SM preparation made a useful system for studying the role of CaM in the brain SM.