FRACTIONATION OF TRITON X-100 SOLUBILIZED PIGEON ERYTHROCYTE MEMBRANE AND RECONSTITUTION INTO VESICLES WITH ATP DEPENDENT CALCIUM ION TRANSPORT ACTIVITY

ATP-dependent (\u2745)Ca(\u272+) transport activity was reconstituted from pigeon erythrocyte membranes solubilized with the nonionic detergent Triton X-100 ( TX-100 ). TX-100 solubilized membrane was fractionated into three peaks by molecular exclusion chromatography with Biogel A1.5M. Ca(\u272+) transport activity could be reconstituted from the sum of the fractions comprising the two lower molecular weight peaks. Reconstitution of activity from these column fractions absolutely required the addition of liposomes (Phosphatidyl choline:Phosphatidyl ethanolamine:Cholesterol:TX-100, 2:2:1:0.625, w/w; added phospholipid:total TX-100 approximately 0.42) and holding several hours at 0(DEGREES)C before removing TX-100. TX-100 was removed by addition of bovine high density lipoprotein which combines avidly with TX-100. Although not absolutely required, reconstitution with a mixture of amino acids, GTP, PEP and inorganic phosphate greatly increased Ca(\u272+) transport activity. These substances were known to increase Ca(\u272+) transport by membrane vesicles. A negative assay protocol was used to locate active material in the Biogel fractions. Successive fractions were deleted from the sum of all fractions comprising the two lower molecular weight peaks. Active material was in the higher molecular weight half of each of these two peaks as indicated by loss of activity upon deletion. Both regions were required. The same activity location was obtained by a supplemental positive assay done by combining limiting amounts of material from the active regions and supplementing these with successive fractions. Both active fractions were heat labile (80(DEGREES)C) and so neither is likely to be only calmodulin. The supplemental positive assays also indicated the presence of inhibitors between the two lower molecular weight peaks and above the higher of these peaks