Calmodulin binding to rat adipocyte plasma membrane: characterization and photoaffinity cross-linking of calmodulin to binding proteins.
[125I]Calmodulin binding to isolated rat adipocyte plasma membranes has been characterized, and the calmodulin binding proteins associated with the membrane have been 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 calcium dependent, requiring 2.2 microM free calcium for half-maximal binding. Total binding was curvilinear with time, plateauing at 30 min. In addition, 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 ethylenediaminetetraacetic acid (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 calcium concentration. The integration of these two techniques provides a powerful tool for studying calmodulin-regulated proteins.
D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D010777:Photochemistry; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D002118:Calcium