LUMENAL AND CYTOPLASMIC-BINDING SITES FOR CALCIUM ON THE CALCIUM-ATPASE OF SARCOPLASMIC-RETICULUM ARE DIFFERENT AND INDEPENDENT

The calcium ATPase of sarcoplasmic reticulum reacts with inorganic pho sphate (P-i) to form phosphoenzyme that can bind two Ca2+ ions from th e lumen of intact vesicles. Therefore, as the concentration of lumenal Ca2+ is increased, the concentration of phosphoenzyme at equilibrium increases; however, it levels off at lower maximal concentrations with decreasing concentrations of P-i. This requires that two Ca2+ ions ca n bind to lumenal binding sites of both the phosphoenzyme and the unph osphorylated enzyme. If lumenal Ca2+ could bind only to the phosphoenz yme, saturating concentrations of lumenal Ca2+ would drive phosphoenzy me formation to completion even at low concentrations of P-i. Phosphor ylation is inhibited by cytoplasmic Ca2+ with K-0.5 = 2.1 and 4 mu M i n the absence and in the presence of 40 mM lumenal Ca2+, respectively. K-0.5 = 4 mu M is much lower than K-0.5 = 70 mu M, which is expected if lumenal Ca2+ could bind only to the phosphoenzyme. Occupancy of the lumenal sites on the unphosphorylated enzyme by Ca2+ does not signifi cantly change the rate constants of k(p) = 220 s(-1) for phosphorylati on by ATP, k(Ca) = 90 s(-1) for dissociation of Ca2+, and k(Mg), = 50 s(-1) for dissociation of Mg2+. We conclude that the calcium ATPase ha s two low-affinity lumenal Ca2+-binding sites that are independent of the high-affinity cytoplasmic Ca2+-binding sites. The results are cons istent with a mechanism of Ca2+ transport in which phosphorylation of the enzyme by ATP drives the translocation of two Ca2+ ions from the h igh-affinity to the low-affinity sites.