Calmodulin binding to rat adipocyte plasma membrane: characterization and photoaffinity crosslinking of calmodulin to binding proteins

Abstract

[125I]Calmodulin binding to isolated rat adipocyte plasma membranes was characterized, and the calmodulin binding proteins associated with the membrane were identified by use of the photoaffinity cross-linker N-hydroxysuccinimidyl 4-azidobenzoate. Total binding of [125I]calmodulin to plasma membranes was assayed by a centrifugation method and found to be Ca dependent, requiring 2.2 .mu.M free Ca for half-maximal binding. Total binding was curvilinear with time, plateauing at 30 min. Calmodulin binding was demonstrated to be both saturable (1700 pmol/mg of membrane protein) and exchangeable. Additional calmodulin binding sites were not produced by further ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N''N''-tetraacetic acid (EGTA) treatment of membranes prepared in the presence of EDTA. Eight specific calmodulin binding protein complexes were identified by use of the photocovalent cross-linking agent. Results obtained with photocovalent cross-linking were similar to those obtained in the total calmodulin binding assays. The formation of the calmodulin binding protein complexes was dependent on time and Ca concentration. The integration of these 2 techniques provides a powerful tool for studying calmodulin-regulated proteins.