Tightly bound calcium of adenosine triphosphatase in sarcoplasmic reticulum from rabbit skeletal muscle.
Sarcoplasmic reticulum vesicles were shown to possess a class of tightly bound calcium ions, inaccessible to the chelator, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid at 0 degrees C or 25 degrees C, amounting to 4.5 nmol/mg of protein (approximately 0.5 mol/mol (Ca2+,Mg2+)-ATPase). The calcium ionophores, A23187 and X537A, induced rapid exchange of tightly bound calcium in the presence of chelator. Chelator alone at 37 degrees C, caused irreversible loss of bound calcium, which correlated with uncoupling of transport from (Ca2+,Mg2+)-ATPase activity. Uncoupling was not accompanied by increased permeability to [14C]inulin. Slow exchange of tightly bound calcium with medium calcium was unaffected by turnover of the ATPase or by tryptic cleavage into 55,000- and 45,000-dalton fragments. Binding studies with labeled calcium suggested that tight binding involves a two-step process: Ca2+ + E in equilibrium K E . Ca2+ leads to E < Ca2+ where E and < Ca2+ represent the ATPase and tightly bound calcium, and K = 1.6 X 10(3) M-1. It is suggested that tightly bound calcium is located in a hydrophobic pocket in, or in close proximity to the ATPase, and, together with tightly bound adenine nucleotides (Aderem, A., McIntosh, D. B., and Berman, M. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3622-03632), is related to the ability of the ATPase to couple hydrolysis of ATP to vectorial transfer of calcium across the membrane.
D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000032:analysis* / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin / Q000494:pharmacology
D000001:Calcimycin; D004533:Egtazic Acid; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium