Abstract
The Ca 2+-pumping ATPase of the plasma membrane of human erythrocytes can be solubilized with nonionic detergents and purified to homogeneity using calmodulin-affinity chromatography. A lipid-free form of the ATPase can be prepared by washing the enzyme bound to the affinity column with detergent. The delipidated ATPase is inactive but can be reactivated by the addition of exogenous phospholipids. The purified ATPase can be reconstituted in phospholipid vesicles by a procedure combining the use of a nonionic detergent with cholate dialysis. The reconstituted vesicles are highly impermeable to ions and accumulate Ca 2+ when energized with ATP. The accumulated Ca 2+ can be released by the addition of Ca 2+ ionophores. The procedure can also be used to isolate and reconstitute the calmodulin-activated Ca 2+-pumping ATPase from other tissues and to study expressed forms of the ATPase.
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