Isolation of a High Affinity Calcium-binding Protein from Sarcoplasmic Reticulum

Seven proteins have been isolated from sarcoplasmic reticulum. These proteins have been identified as an ATPase, a series of water soluble, acidic proteins of molecular weights 55,000, 46,500, 38,000, 33,000, and 20,000, and a proteolipid. The interactions of the acidic proteins with Ca2+ have been measured at pH 7.5 in the presence and absence of 0.1 m KCl. The protein of molecular weight 55,000 is the only protein with high affinity for Ca2+. In the presence of 0.1 m KCl it binds 16 to 22 nmoles of Ca2+ per mg (about 1 mole of Ca2+ with a dissociation constant between 2.5 and 4 µm. It also binds several hundred nanomoles of Ca2+ with a dissociation constant greater than 5,000 µm. In the absence of KCl the protein of molecular weight 55,000 displays the high affinity Ca2+ binding site and sites which bind about 460 nmoles of Ca2+ per mg with a dissociation constant of about 120 µm. In the presence of 0.1 m KCl, calsequestrin (mol wt 46,500) does not show high affinity Ca2+ binding but binds about 850 nmoles of Ca2+ per mg with a dissociation constant of about 800 µm. In the absence of KCl, calsequestrin binds about 900 nmoles of Ca2+ per mg with a dissociation constant of about 60 µm. In the absence of KCl the acidic proteins with molecular weights between 20,000 and 38,000 bind from 900 to 1,100 nmoles of Ca2+ per mg with dissociation constants in the range of 115 to 150 µm. Apparent high affinity Ca2+ binding is observed in these fractions in the absence of KCl but, in the presence of 0.1 m KCl, both the apparent high affinity Ca2+ binding and the low affinity binding are greatly reduced.