title: The effect of calmodulin on the interaction of carbodiimides with the purified human erythrocyte (Ca2+ + Mg2+)-ATPase.
abstract: The activity of the solubilized and purified (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes was inhibited by N,N'-dicyclohexylcarbodiimide in a concentration-dependent manner. The carbodiimide prevented formation of the phosphorylated intermediate during the catalytic cycle of the enzyme. Treatment of the enzyme with N,N'-dicyclohexyl[14C]carbodiimide resulted in the formation of a 14C-labelled polypeptide corresponding to the enzyme monomer (molecular weight 136,000). The tryptic fragmentation of this 14C-labelled enzyme resulted in the formation of three major 14C-labelled fragments with molecular weights of 58,000, 36,500 and 23,000, the latter two probably representing transmembrane and calmodulin-binding domains of the enzyme, respectively. In the absence of calmodulin, 6.7 molecules of N,N'-dicyclohexyl[14C]carbodiimide covalently bound to each molecule of Ca2+-ATPase; in the presence of calmodulin, the number of molecules of carbodiimide bound was 13.1. The binding of N,N'-dicyclohexylcarbodiimide to the (Ca2+ + Mg2+)-ATPase greatly reduced its ability to bind to a calmodulin-agarose gel.
authors.affiliation: Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
authors.forename: O O
authors.initials: OO
authors.lastname: Olorunsogo
authors.forename: A
authors.initials: A
authors.lastname: Villalobo
authors.forename: K K
authors.initials: KK
authors.lastname: Wang
authors.forename: B D
authors.initials: BD
authors.lastname: Roufogalis
mesh_terms: D017301:Ca(2+) Mg(2+)-ATPase / Q000097:blood* / Q000302:isolation & purification; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000302:isolation & purification; D002147:Calmodulin / Q000378:metabolism*; D002234:Carbodiimides / Q000494:pharmacology*; D002846:Chromatography, Affinity; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding
chemical_list: D002147:Calmodulin; D002234:Carbodiimides; D004024:Dicyclohexylcarbodiimide; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases