The Biochemical journal. vol:263 issue:3 pages:687-94
The aim of the present work was to investigate the stimulation of the plasma-membrane Ca2+-transporting ATPase by negatively charged phospholipids. The Ca2+-transporting ATPase was purified from pig stomach smooth muscle and from pig erythrocytes, and was reactivated with phosphatidylcholine (PC) in the presence and absence of negatively charged phospholipids. The substitution of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA) or phosphatidylserine (PS) for PC induced profound changes in the Vmax, the K0.5 and the Hill coefficient of the Ca2+-activation curves for both ATPases. Low concentrations of each of the negatively charged phospholipids increased the Vmax., but high ratios of PIP, PIP2 or PA to PC decreased this parameter. PI, PA and PS increased the Vmax. of the erythrocyte enzyme to a larger extent than that of the smooth-muscle enzyme. This difference was less pronounced for PIP and absent for PIP2. PI (greater than 20% PC substituted), PIP, PIP2, PA and PS all increased the affinity of the two Ca2+-transporting ATPases for Ca2+ in the following order of potency: PIP2 greater than PIP greater than PI approximately PS approximately PA. PI, PA and PS increased the Ca2+ affinity of the smooth-muscle enzyme more than that of the erythrocyte enzyme; this difference was less pronounced for PIP and absent for PIP2. Even in the presence of calmodulin, all of the negatively charged phospholipids were still able to increase the Vmax. of the erythrocyte enzyme, whereas only PIP and PIP2 increased the affinity for Ca2+. The effect of PI at low concentrations (less than 20%) on the erythrocyte enzyme was peculiar in that it caused a decrease in the Ca2+ affinity instead of an increase. This effect was not observed for the smooth-muscle enzyme. All of the negatively charged phospholipids slightly increased the Hill coefficient for Ca2+ of both ATPases, and this effect was additive to that of calmodulin. The stimulation of the erythrocyte enzyme exhibited positive co-operativity towards PI and PIP, whereas that of the smooth-muscle enzyme did not. It is concluded (1) that there is a correlation between the number of negative charges on the phospholipids (PIP2 greater than PIP greater than PA approximately PI approximately PS) and the magnitude of their effect on the Vmax. and the K0.5 for Ca2+, and (2) that the action of the lipids on the smooth-muscle enzyme differs from that on the erythrocyte enzyme, indicating that these two Ca2+-transporting ATPases are not the same.