Ionophorous properties of the 20,000-dalton fragment of (Ca2++Mg2+)-ATPase in phosphatidylcholine: Cholesterol membranes

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Abstract
The purified 20,000-dalton fragment of sarcoplasmic reticulum (Ca2++Mg2+)-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 Ca2+-selective ionophoric activity. The Ca2+-ionophoric fragment has been purified by either SDS-column chromatography or SDS-preparative gel electrophoresis. The Ca2+-ionophoric fragment has been subjected 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>PMn. This selectivity sequence is the same as that for the intact (Ca2+ +Mg2+)-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 thatPMn>PMg. This and other data indicate that the 20,000-dalton fragment is the site containing the Ca2+-ionophoric activity of the (Ca2++Mg2+)-ATPase.