J. Biol. Chem., Vol. 255, Issue 23, 11351-11356, Dec, 1980
Tightly bound calcium of adenosine triphosphatase in sarcoplasmic reticulum from rabbit skeletal muscle
EM Diamond, KB Norton, DB McIntosh and MC Berman
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.