Occlusion of Divalent Cations in the Phosphorylated Calcium Pump of Sarcoplasmic Reticulum

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Abstract
The Ca pump of rabbit muscle sarcoplasmic reticulum possesses high-affinity Ca-binding and ATP-binding sites. At 0.degree. C, pH 6.8 and in the absence of Ca, about 3.5 nmol/mg of high-affinity ATP-binding sites are titrated with a dissociation constant, Kd, of 5 .mu.M. In the presence of Ca2+, ATP phosphorylates the enzyme at a much lower concentration: K1/2 = 100 nM. In the absence of ATP, Ca ions reversibly bind to the high-affinity Ca sites (6.5 nmol/mg). Phosphorylation of the enzyme in the presence of Ca leads to the immediate occlusion of the Ca ions bound to the high-affinity sites. Two moles of Ca are occluded per mole of phosphoenzyme formed. Occlusion can be reversed by ADP. Transport is a slower process which occurs in the presence of Mg2+ at the same rate as the spontaneous decay of the phosphoenzyme. Experiments performed in the absence of Mg reveal another divalent cation binding site which is probably directly involved in ATP and Pi binding. The nature of the cation bound to this site determines the stability and ADP-sensitivity of the phoshoenzyme.