Calcium ATPase of sarcoplasmic reticulum has four binding sites for calcium.

The calcium-transporting ATPase of sarcoplasmic reticulum is known to bind two Ca2+ ions from the cytoplasm to the free enzyme and two Ca2+ ions from the lumen to the phosphoenzyme. The concentration of phosphoenzyme formed at equilibrium from Pi and Mg2+ increases with increasing concentration of calcium in the lumen, which binds to the phosphoenzyme to form Ca2.E approximately P.Mg. However, at subsaturating concentrations of Mg2+ increasing the concentration of lumenal Ca2+ does not drive phosphoenzyme formation to completion. The maximal levels of phosphoenzyme that are formed at saturating concentrations of lumenal Ca2+ increase with increasing concentrations of Mg2+. This result requires that Ca2+ can bind to low-affinity lumenal sites on both the free enzyme and the phosphoenzyme, as well as to the high-affinity cytoplasmic calcium-binding sites. If there were no lumenal binding sites for Ca2+ on the free enzyme, high concentrations of lumenal Ca2+ would convert all of the enzyme to the same maximal concentration of Ca2.E approximately P.Mg at subsaturating concentrations of Mg2+ and Pi. We conclude that there are two low-affinity lumenal sites as well as two high-affinity cytoplasmic sites for Ca2+ on the free enzyme. Phosphorylation by ATP results in translocation of Ca2+ from the high-affinity to the low-affinity sites.