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 KC1 it binds 16 to 22 nmoles of Ca2+ per mg (about 1 mole of Ca2+ per mole) with a dissociation constant between 2.5 and 4 pM. It also binds several hundred nanomoles of Ca2+ with a dissociation constant greater than 5,000 FM. In the absence of KC1 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 pM. 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 pM. In the absence of KCl, calsequestrin binds about 900 nmoles of Ca2f per mg with a dissociation constant of about 60 pM. In the absence of KC1 the acidic proteins with molecular weights between 20,000 and 38,000 bind from 900 to 1,100 nmoles of Ca’+ per mg with dissociation constants in the range of 115 to 150 pM. Apparent high affinity Ca 2+ binding is observed in these fractions in the absence of KC1 but, in the presence of 0.1 M KCl, both the apparent high affinity Ca2+ binding and the low affinity binding are greatly reduced.