Cooperative calcium binding and ATPase activation in sarcoplasmic reticulum vesicles.

High affinity calcium binding which is involved in ATPase activation was measured following equilibration of sarcoplasmic reticulum vesicles with Ca2+ buffers in chromatography columns. Binding occurred with ir,,, = 2.3 X lo6 M” and with a maximal level of 8 to 10 m o l per mg of protein. This value corresponds to 2 calcium ions per ATPase phosphorylation site, since the maximal level of phosphorylated enzyme intermediate obtained in the presence of ATP was 4 to 5 nmol per mg of protein. Analysis of calcium binding as a function of Ca” concentration demonstrates a cooperative mechanism including binding to a first site, followed by development of higher affinity and binding to a second site. Accordingly, the best fit of the equilibrium binding curve requires two constants, with a 2 orders of magnitude increase in the second as compared to the first constant. Scatchard and Hill plots also demonstrate a highly cooperative ( n ~ =; 1.82) binding mechanism. High affinity calcium binding is accompanied by a protein conformational change which is revealed by EPR spectroscopy of spin-labeled preparations and is consistent with the analysis developed for the equilibrium binding data. It is apparent that such a conformational change is an integral part of the cooperative binding mechanism and of the enzyme activation following calcium binding, Kinetic experiments on enzyme phosphorylation upon addition of ATP to enzyme preincubated with Ca2+, as compared to addition of ATP and Ca’+ to enzyme deprived of Ca2+, revealed that the Ca2+-induced enzyme activation is a slow step which is influenced by substrate (e.g. ATP) binding. In conclusion, equilib~um and kinetic analysis indicate the following mechanism for high affinity calcium binding and ATPase activation: E + Caa+ .+ E.Ca + E’oCa + Ca” -+ E”-Ca2.