Phospholipids are necessary for calmodulin-stimulated activation of the Ca(2+)-ATPase of erythrocytes.
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+.
Gazzotti
P
P
Laboratory of Biochemistry III, Swiss Federal Institute of Technology (ETH), Zürich.
Gloor-Amrein
M
M
Adebayo
R
R
D000252:Calcium-Transporting ATPases / Q000097:blood*; D002147:Calmodulin / Q000502:physiology; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D017830:Octoxynol; D010743:Phospholipids / Q000502:physiology*
D002147:Calmodulin; D010743:Phospholipids; D017830:Octoxynol; D000252:Calcium-Transporting ATPases