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Plasma membrane Ca2+-ATPase activity diminishes by about 50% in red blood cells during preeclampsia. We investigated whether the number of Ca2+-ATPase molecules is modified in red cell membranes from preeclamptic pregnant women by measuring the specific phosphorylated intermediate of this enzyme. Also, we isolated the Ca2+-ATPase protein from both normotensive and preeclamptic pregnant women and estimated its molecular weight, and its cross-reactions with specific polyclonal and monoclonal (5F10) antibodies against it. We measured the Ca2+-ATPase activity in a purified state and the effect of known modulators of this ATPase. It was found that the phosphorylated intermediate associated with PMCA is similar for red cell ghosts from normotensive and preeclamptic women, suggesting a similar number of ATPase molecules in these membranes. The molecular weight of the Ca2+-ATPase is around 140 kDa for both normotensive and preeclamptic membranes, and its cross-reactions with specific antibodies is similar, suggesting that the protein structure remains intact in preeclampsia. Calmodulin, ethanol, or both calmodulin plus ethanol, stimulated the Ca2+-ATPase activity to the same extent for both normotensive and preeclamptic preparations. Our results showed that the reduced Ca2+-ATPase activity of the red cell membranes from preeclamptic women is not associated with a defective enzyme, but rather with a high level of lipid peroxidation.

The plasma membrane Ca(2+)-ATPase from human erythrocytes can be stimulated by different treatments such as addition of calmodulin or acidic phospholipids and controlled proteolysis. In this report we show that short chain alkyl alcohols also stimulated this enzyme. At 5% (v/v) ethanol, the maximal velocity of the enzyme was about 2.4-fold higher than in the control, and thus, was also higher than the maximal velocity obtained in the presence of calmodulin (about 2-fold). When ethanol and calmodulin were present simultaneously, the stimulatory effect was additive (3.4-fold stimulation). On the other hand, the stimulatory effect of ethanol was preserved after treatment of the enzyme with trypsin to stimulate the Ca(2+)-ATPase and render it independent of calmodulin, thus suggesting that the interaction of ethanol and calmodulin with the Ca(2+)-ATPase occurred through a different mechanism. Other short chain alkyl alcohols (methanol, n-propanol and n-butanol) stimulated the Ca(2+)-ATPase activity to the same extent than ethanol but with different efficacy. Thus, the larger the carbon number, the lower the concentration needed to get the same maximal stimulation. Ethanol also increased the affinity of the enzyme for ATP to a larger extent and additively, when compared to calmodulin. All the effects of ethanol mentioned above were identically observed on the membrane-bound enzyme (i.e., erythrocyte ghosts) ruling out any effect of the alcohols attributable to the solubilized purified enzyme. Furthermore, Ca2+ transport by inside-out vesicles was also stimulated by ethanol, showing both the same concentration-dependence as the Ca(2+)-ATPase activity and the additive effect observed when calmodulin was also present. The stimulatory effect of ethanol was significant at pharmacological concentrations, thus suggesting potential implications of toxicological relevance.

A subcellular fraction highly enriched in plasma membrane vesicles was prepared from Leishmania promastigotes. This fraction showed (Ca2+ + Mg2+)-ATPase activity. This, however, represented a small fraction (about 25%) of the overall ATPase activity. The Ca2(+)-ATPase showed general characteristics common to plasma membrane ATPases involved in Ca2+ transport. Thus, the Ca2(+)-ATPase was activated by Ca2+ with a high affinity (Km about 0.7 microM), saturating at about 5 microM Ca2+. Furthermore, it was stimulated by calmodulin (about 70-80% with 5 micrograms/ml) and almost fully inhibited by trifluoperazine (100 microM). The above vesicles accumulated Ca2+ against a concentration gradient and released it after the addition of A23187, as shown independently by 45Ca2+ and Arsenazo III studies. The transport mechanism showed the same kinetics parameters as described for the enzyme, indicating a single molecular entity. In addition, Ca2(+)-ATPase activity and Ca2+ uptake were completely inhibited by vanadate (20 microM), indicating that an E1-E2 type mechanism is involved. The results clearly demonstrate the presence of a Ca2+ pump in the plasma membrane of Leishmania which is capable of maintaining a low cytoplasmic Ca2+ concentration.

The Ca2(+)-ATPase of the erythrocyte plasma membrane can be activated by calmodulin, acidic phospholipids, limited proteolysis and self-association. Recently, it has been shown that different organic solvents increase both the Vmax and the Ca2+ affinity of the enzyme (Benaim, G. and De Meis, L. (1989) FEBS Lett. 244, 484-486). In this report the effects of calmodulin and dimethyl sulfoxide (20%, v/v) on the Ca2(+)-ATPase are compared. Dimethyl sulfoxide also elicits the appearance of the low-affinity binding site, which in this enzyme is strictly dependent on calmodulin. Dimethyl sulfoxide increases the Ca2+ affinity of the enzyme in a manner similar to that observed with the use of calmodulin and of acidic phospholipids. This was tested using both native and partially trypsinized ATPase. When activated by calmodulin the enzyme is inhibited by compound 48/80, trifluoperazine and calmidazolium. When activated by dimethyl sulfoxide the enzyme is still inhibited by calmidazolium but is no longer inhibited by either compound 48/80 or trifluoperazine. Activation of the ATPase promoted by either calmodulin or dimethyl sulfoxide is abolished when the Ca2+ concentration is raised from 10 microM to 2 mM. The effect of dimethyl sulfoxide is also abolished by 20 mM Pi. In the presence of 1 to 10 mM Ca2+ the ATPase catalyzes an ATP in equilibrium Pi exchange. The rate of exchange increases several fold when dimethyl sulfoxide is included in the assay medium.