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The plasma membrane Ca(2+)-ATPase (PMCA) plays a key role in the regulation of the intracellular Ca(2+) concentration. Ethanol stimulates this Ca(2+) pump in an isoform-specific manner. On search for a physiological molecule that could mimic the effect of ethanol, we have previously demonstrated that some sphingolipids containing free "hydroxyl" groups, like ceramide, are able to stimulate the PMCA. Since diacylglycerol (DAG) structurally shares some characteristics with ceramide, we evaluate its effect on the PMCA. We demonstrated that DAG is a potent stimulator of this enzyme. The activation induced is additive to that produced by calmodulin, protein-kinase C and ethanol, which implies that DAG interacts with the PMCA through a different mechanism. Additionally, by different fluorescent approaches, we demonstrated a direct binding between PMCA and DAG. The results obtained in this work strongly suggest that DAG is a novel effector of the PMCA, acting by a direct interaction.

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 is a key enzyme in the regulation of the intracellular Ca(2+) concentration. On the other hand, sphingolipids have been recognized recently as important second messengers, acting in many systems in combination with Ca(2+). In view of the fact that the Ca(2+)-ATPase is stimulated by ethanol, and since sphingolipids possess free hydroxy groups, we decided to study the possible effect of ceramide and sphingosine on this calcium pump. Here we show that ceramide stimulates the Ca(2+)-ATPase in a dose-dependent manner and additively to the activation observed in the presence of calmodulin or ethanol, when compared with any of these effectors added alone. Ceramide affects both the affinity for Ca(2+) and the V(max) of the enzyme. Furthermore, this second messenger also stimulates Ca(2+) transport in inside-out plasma-membrane vesicles from erythro cytes. Conversely, sphingosine, which is reported to act in many systems antagonistically with ceramide, showed an inhibitory effect on Ca(2+)-ATPase activity. This inhibition was also observed on the calmodulin-stimulated enzyme. These results, taken together, suggest that ceramide and sphingosine act antagonistically on the plasma-membrane Ca(2+)-ATPase. This is in accordance with the frequently reported opposite effect of these sphingolipids on intracellular Ca(2+) concentration.

Phosphatidylethanol is formed by "transphosphatidylation' of phospholipids with ethanol catalysed by phospholipase D and can be accumulated in the plasma membrane of mammalian cells after treatment of animals with ethanol. In the present work we show that phosphatidylalcohols, such as phosphatidylethanol and phosphatidylbutanol, produced a twofold stimulation of the Ca(2+)-ATPase activity of human erythrocytes. This stimulation occurs with the purified, solubilized enzyme as well as with ghost preparations, where the enzyme is in its natural lipidic environment and is different to that obtained with other acidic phospholipids such as phosphatidylserine. Addition of either phosphatidylserine, phosphatidylethanol or phosphatidylbutanol to the purified Ca(2+)-ATPase, or to ghosts preparations, increased the affinity of the enzyme for Ca2+ and the maximal velocity of the reaction as compared with controls in the absence of acidic phospholipids. However, in contrast with what occurs with phosphatidylserine, simultaneous addition of phosphatidyl-alcohols and calmodulin increased the affinity of the enzyme for Ca2+ to a greater extent than each added separately. When ethanol was added to either the purified erythrocyte Ca(2+)-ATPase or to erythrocyte-ghost preparations in the presence of acidic phospholipids, an additive effect was observed. There was an increase in the affinity for Ca2+ and in the maximal velocity of the reaction, well above the values obtained with ethanol or with the acidic phospholipids tested separately. These findings could have pharmacological importance. It is conceivable that the decrease in the intracellular Ca(2+) concentration that has been reported in erythrocytes as a result of ethanol intoxication could be due to the stimulation of the Ca(2+)-ATPase by the accumulated phosphatidylethanol, to a direct effect of ethanol on the enzyme or to an additive combination of both.

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.

Subunit interactions in the Ca(2+)-ATPase from erythrocyte plasma membranes were investigated through a combination of fluorescence spectroscopy and high-pressure techniques. Application of hydrostatic pressure in the range of 1 bar to 2.4 kbar promoted full dissociation of the ATPase, as revealed by spectral shifts of the intrinsic fluorescence emission and by changes in the fluorescence polarization of dansyl-conjugated ATPase. Pressure dissociation of the ATPase displayed a dependence on protein concentration compatible with dissociation of a dimer. Calculated from pressure-dissociation curves, the standard volume change dV0 for the association of subunits was 43-50 ml/mol and K0, the dissociation constant at atmospheric pressure, was 6-9 x 10(-8) M. Addition of Ca2+ stabilized the dimeric ATPase structure against pressure dissociation, whereas addition of vanadate facilitated dissociation by pressure. These results suggest that intersubunit interactions depend on the equilibrium between the two major conformational states E1 and E2 of the ATPase. Addition of calmodulin in the presence of Ca2+ had no additional effect when compared to that observed in the presence of Ca2+ alone. This finding is interpreted in terms of the mechanism of calmodulin activation of ATPase catalysis.

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.

The purified Ca2+-pumping ATPase of the erythrocyte membrane has been exposed to trypsin at 37 degrees C, in the presence of different effectors of its activity. The control proteolytic pattern is characterized by a number of transient and of limit polypeptides (Zurini, M., Krebs, J., Penniston, J. T., and Carafoli, E. (1984) J. Biol. Chem. 259, 618-627). The effectors influence the pattern in the Mr region 90,000-76,000, which contains the calmodulin binding domain and the active site of the enzyme. In this region, polypeptides of 90, 85, 81, and 76 kDa are clearly visible in the controls. 1) Calmodulin plus Ca2+ induces the faster disappearance of the 90-kDa product and the relative accumulation of the 85-kDa with respect to the 81-kDa polypeptide. 2) Vanadate plus Mg2+ also accelerates the disappearance of the 90-kDa product. However, they induce the relative accumulation of the 81-kDa polypeptide. 3) Linoleic acid, which stimulates the activity of the enzyme to the same levels obtained with calmodulin, greatly accelerates the rate of trypsin proteolysis, causing the virtual disappearance of all polypeptides in the 90-76-kDa region. 4) The 81-kDa polypeptide has maximal ATPase activity and is insensitive to calmodulin; the 85-kDa polypeptide has lower ATPase activity and binds calmodulin, but is not stimulated (or is stimulated only negligibly) by the activator.

The purified Ca2+ ATPase of the erythrocyte plasma membrane has been submitted to controlled trypsin proteolysis under conditions that favor either its (putative) E1 or E2 configurations. The former configuration has been forced by treating the enzyme with Ca2+-saturated calmodulin, the latter with vanadate and Mg2+. The E1 conformation leads to the accumulation of a polypeptide of Mr 85 KDa which still binds calmodulin, the E2 conformation to the accumulation of one of Mr 81 KDa which does not. Both fragments arise from the hydrolysis of a transient 90 KDa product which has Ca2+-calmodulin dependent ATPase activity, and which retains the ability to pump Ca2+ in reconstituted liposomes. Highly enriched preparations of the 85 and 81 KDa fragments have been obtained and reconstituted into liposomes. The former has limited ATPase and Ca2+ transport ability and is not stimulated by calmodulin. The latter has much higher ATPase and Ca2+ transport activity. It is proposed that the Ca2+ pumping ATPase of erythrocytes plasma membrane contains a 9 KDa domain which is essential for the interaction of the enzyme with calmodulin and for the full expression of the hydrolytic and transport activity. This putative 9 KDa sequence contains a 4 KDa "inhibitory" domain which limits the activity of the ATPase. In the presence of this 4 KDa sequence, i.e., when the enzyme is degraded to the 85 KDa product, calmodulin can still be bound, but no longer stimulates ATPase and Ca2+ transport.

In this report it is shown that organic solvents mimic the stimulatory effects of calmodulin and acidic phospholipids on the erythrocyte plasma membrane Ca2+-ATPase. The solvents used were dimethyl sulfoxide (20%, v/v), glycerol (20% v/v), ethylene glycol (20%, v/v) and polyethylene glycol (Mr 6000-8000) (10%, w/v). These solvents increased both the affinity for Ca2+ and the turnover number of the enzyme. The increase in Ca2+ affinity is additive to that achieved with calmodulin. The calcium cooperativity observed in the presence of calmodulin disappears after the addition of dimethyl sulfoxide to the medium. The present data support the proposal that activation of the erythrocyte plasma membrane Ca2+-ATPase is promoted by hydrophobic interactions along the enzyme molecule.