249 Biochimica et Biophysica Acta, "555 (1979) 249--258 © Elsevier/North-Holland Biomedical Press BBA 78444 ACTIVE Ca 2+ TRANSPORT BY VESICLES RECONSTITUTED FROM TRITON X.100-SOLUBILIZED PIGEON ERYTHROCYTE MEMBRANE WANG K. YEUNG *, GARY WEISMAN and GEORGE A. VIDAVER ** Department of Chemistry, University of Nebraska at Lincoln, Lincoln, NE 68588 (U.S.A.) (Received March 15th, 1979) Key words: Ca 2+ transport; Membrane reconstruction; Triton X-I O0 solubilization; (Pigeon ery throcy te mere brahe) Summary Pigeon erythrocyte membrane was solubilized partially, but relatively unselectively by Triton X-100. Vesicles were reconstituted from mixtures of Triton-solubilized membrane and lipid (phosphatidylcholine plus phosphatidyl- ethanolamine plus cholesterol) by addition of bovine high-density lipoprotein. This efficiently removed the Triton X-100. Sodium dodecyl sulfate-polyacryl- amide gel electropherograms of reconstituted vesicles showed band patterns resembling those of the original membrane. The reconstituted vesicles showed ATP-dependent active accumulation of 4SCa2+. ATP-dependent 4SCa:* uptake by the reconstituted vesicles resembled the corresponding activity of the original membrane vesicles; in both preparations the Ca :÷ uptake rate depended on the square of the Ca ~÷ concentration and had similar [Ca2+]l/2 values, 0.16/AVI and 0.18 pAM, respectively. Introduction Several procedures are available for reconstituting transport-active vesicles from detergent-solubilized membranes; for example, Refs. 1--9. No procedure is quick, gentle and efficient in removing the detergent. An ideal general proce- dure should also be unselective for both solubilization and reconstitution. * Present address: Division of Molecular and Cellular Biology, National Jewish Hospital and Research Center, 3800 East Colfax Ave., Denver, CO 80206, U.S.A. ** To whom reprint requests should be sent. Abbreviations: SDS, sodium dodeeyl sulfate; HDL, bovine high-density lipoprotein, d = 1.17, prepared and density calculated as described in Ref. 10 and footnote p. urn; EGTA, ethylene glycol bis(~3-amino- ethyl ether)-N, Nt-tetraaeetic acid.