Reconstitution of the Ca2+-transport system of human erythrocytes.
The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte 'ghosts' was solubilized and reconstituted to form membranous vesicles capable of energized Ca2+ accumulation. The erythrocyte 'ghosts' for this purpose were prepared by using isoosmotic freeze-haemolysis in the presence of Tween 20 and proteinase inhibitors to stabilize the preparation. The reconstitution procedure is similar to that developed by Meissner & Fleischer [(1974) J. Biol. Chem. 249, 302-309] for skeletal-muscle sarcoplasmic-reticulum in that: (1) deoxycholate is used for the solubilization of the membrane; (2) controlled dialysis at near room temperature, rather than 0 degree C, is required in order to obtain a functional preparation capable of Ca2+ accumulation; and (3) membrane vesicles can be reassembled with protein/lipid ratio (approx. 60% protein and 40% lipid) similar to that of the original membrane.
D001692:Biological Transport; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000097:blood*; D003840:Deoxycholic Acid; D003956:Dialysis; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D008722:Methods; D011136:Polysorbates
D011136:Polysorbates; D003840:Deoxycholic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium