ABSTRACT
Treatment of red cell ghosts with increasing concentrations of the non-ionic detergent Triton X-100 caused a progressive loss of Ca(2+)-ATPase activity. Both the basal activity and the calmodulin-stimulated activity were affected and could be partially restored by acidic phospholipids. Lipid-free Ca(2+)-ATPase was prepared from solubilized ghosts by calmodulin affinity chromatography and extensive washing of the column with detergent to remove the endogenous phospholipids associated with the enzyme. The phospholipid-free, solubilized Ca(2+)-ATPase had very low activity and was not activated by calmodulin. The tryptic proteolytic pattern of the delipidated ATPase differed from the pattern of the phospholipid-associated enzyme, indicating that the delipidation had caused conformational changes. The activity was fully restored by phosphatidylserine, but was only partially restored by phosphatidylcholine. The phosphatidylcholine-activated enzyme was restored to maximal activity in the presence of calmodulin. The delipidated ATPase could be reconstituted in soybean lipid vesicles and was able to actively transport Ca2+.
