J. Membrane Biol. 43, 227-242 (1978) Ionophorous Properties of the 20,000-Dalton Fragment of (Ca 2§ + Mg2+)-ATPase in Phosphatidylcholine: Cholesterol Membranes Adil E. Shamoo Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Received 20 March 1978; revised 30 May 1978 Summary. The purified 20,000-dalton fragment of sarcoplasmic reticulum (Ca 2+ +MgZ+)-ATPase has been shown by us (A.E. Shamoo, T.E. Ryan, P.S. Stewart, D.H. MacLennan, 1976. J. Biol. Chem. 251:4147) to have CaZ+-selective ionophoric activity. The CaZ+-ionophoric fragment has been purified by either SDS-column chromatography or SDS-preparative gel electrophoresis. The Ca2+-ionophoric fragment has been sub- jected to prolonged dialysis to insure the removal of bound SDS from the fragment. The selectivity sequence of this fragment in black lipid membranes (BLM) formed from either oxidized cholesterol or phosphatidylcholine/cholesterol is the same, PBa> Pca > Psr > PMg > PNn" This selectivity sequence is the same as that for the intact (Ca 2+ + Mg 2 +)-ATPase. Treatment of the fragment with cholate to absolutely insure the removal of bound SDS resulted in the fragment having a selectivity sequence as above except that P,~ > PMg. This and other data indicate that the 20,000-dalton fragment is the site contain- ing the CaZ+-ionophoric activity of the (Ca2+ + Mg2+)-ATPase. (Ca 2 ++ Mg 2 +)-ATPase 1 is known to be the primary "pump" protein for translocation of Ca 2 + into rabbit white skeletal muscle sarcoplasmic retic- ulum (Racker, 1972; 1973). The enzyme has been reconstituted into artificial vesicles following its purification. These vesicles have been shown to catalyze ATP-dependent Ca 2+ transport. The reconstitution data and other mounting evidence indicating that membrane-bound ATPases contain the entire mechanism necessary for ion transportation prompted our laboratory to look for and find an ion-bearing and ion-translocating site more commonly known as an ionophoric site (Shamoo & Ryan, 1975). In the case of the (Ca z+ +MgZ+)-ATPase molecule, we were able to show that the Ca 2 + ionophorous property resides as part of the intact 1 Abbreviations: (Ca 2 + + Mg 2 +)-ATPase = (Ca z + + Mg 2 +)-dependent adenosine-triphos- phase. 0022-2631/78/0043-0227 $3.20 9 Springer-Verlag New York Inc. 1978