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 phosphate (Pi) to form phosphoenzyme that can bind two Ca2+ ions from the 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 Pi. This requires that two Ca2+ ions can bind to lumenal binding sites of both the phosphoenzyme and the unphosphorylated enzyme. If lumenal Ca2+ could bind only to the phosphoenzyme, saturating concentrations of lumenal Ca2+ would drive phosphoenzyme formation to completion even at low concentrations of Pi. Phosphorylation is inhibited by cytoplasmic Ca2+ with K0.5 = 2.1 and 4 microM in the absence and in the presence of 40 mM lumenal Ca2+, respectively. K0.5 = 4 microM is much lower than K0.5 = 70 microM, 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 significantly change the rate constants of kp = 220 s-1 for phosphorylation by ATP, kCa = 90 s-1 for dissociation of Ca2+, and kMg = 50 s-1 for dissociation of Mg2+. We conclude that the calcium ATPase has two low-affinity lumenal Ca(2+)-binding sites that are independent of the high-affinity cytoplasmic Ca(2+)-binding sites. The results are consistent with a mechanism of Ca2+ transport in which phosphorylation of the enzyme by ATP drives the translocation of two Ca2+ ions from the high-affinity to the low-affinity sites.
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
J
J
Myung
W P
WP
Jencks
D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure
D010710:Phosphates; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium