Thermal analysis of the plasma membrane Ca2+-ATPase

The plasma membrane Ca²⁺-ATPase is a well known enzyme in eucaryotes able to extrude calcium to the extracellular space in order to restore intracellular calcium to very low levels. This ATPase needs plasma membrane lipids such as acidic phospholipids in order to maintain its activity. In this study, we investigated the role that calcium and cholesterol play on the thermal stability of the Ca²⁺-ATPase isolated from cardiac sarcolemma and erythrocyte membranes. Calcium showed a stabilizing and protective effect when the enzyme was exposed to high temperatures. This stabilizing effect showed by calcium was potentiated in the presence of cholesterol. These protection effects were reflected on several thermodynamic parameters such as T₅₀, H_vh and apparent G, indicating that calcium might induce a conformational change stabilized in the presence of cholesterol that confers enzyme thermostability. The effect shown by cholesterol on H_vh and apparent H open the possibility that this lipid decreases cooperativity during the induced transition. Despite that a binding site for cholesterol has not been identified in the plasma membrane Ca²⁺-ATPase, our results supports the proposal that this lipid interacts with the enzyme in a direct fash     

Erythrocyte cytosolic free Ca2+ and plasma membrane Ca2+-ATPase activity in cystic fibrosis

The properties of the Ca2+, Mg2+-ATPase of erythrocyte membranes from patients with cystic fibrosis (CF) were extensively compared to that of healthy controls. Following removal of an endogenous membrane inhibitor of the ATPase, activation of the enzyme by Ca2+, calmodulin, limited tryptic digestion or oleic acid, as well as inhibition by trifluoperazine, were studied. The only properties found to be significantly different (CF cells vs controls) were calmodulin-stimulated peak activity (90 vs 101, P less than 0.02) and trypsin-activated peak activity (92 vs 102, P less than 0.02). No significant difference could be measured in the steady-state Ca2+-dependent phosphorylation of CF and control erythrocyte membranes indicating similar numbers of enzyme molecules per cell. The functional state of Ca2+ homeostasis in intact erythrocytes was investigated by measuring the resting cytosolic free Ca2+ levels using quin-2. Both CF and control erythrocytes maintained cytosolic free Ca2+ between 20 to 30 nM. Addition of 50 uM trifluoperazine resulted in an increase in erythrocyte cytosolic free Ca2+ to about 50 nM in both CF and control cells. Estimates of erythrocyte membrane permeability using the steady-state uptake of 45Ca into intact erythrocytes revealed no differences between CF and control cells. These results confirm that there is a small decrease in the calmodulin-stimulated activity of the erythrocyte Ca2+, Mg2+-ATPase in CF. However, this deficit is apparently not large enough to impair the ability of the CF erythrocyte to maintain normal resting levels of cytosolic free Ca2+.     

Phosphatidylserine externalization in caveolae inhibits Ca2+ efflux through plasma membrane Ca2+-ATPase in ECV304

It has been evidenced that plasma membrane Ca(2+)-ATPase (PMCA) is localized at caveolae. However, the caveolar function of PMCA in living cells has never been demonstrated. In the present study, PMCA is exclusively localized at caveolae from ECV 304 cells demonstrated by sucrose gradient fractionation and the co-localization of PMCA with caveolin-1 was visualized by confocal microscopy. We found that PMCA is the main mechanism involved in Ca(2+) efflux in ECV 304 cells. Treatment of cells with MbetaCD to disrupt caveolae significantly reduced the Ca(2+) efflux, and the rate of decay is 4.45+/-0.14 min(-1) in the absence of MbetaCD and 1.99+/-0.038 min(-1) in the presence of MbetaCD. Moreover, the replenishment of cholesterol restored the reduction of the PMCA-mediated Ca(2+) efflux in the presence of MbetaCD. Consistent with Ca(2+) efflux in living cells, the activity of the reconstituted PMCA in membranes extracted from cells in vitro was decreased in the presence of MbetaCD. It was found that phosphatidylserine, which is normally in the inner leaflet of plasma membranes and is able to stimulate PMCA was relatively enriched in caveolae. Importantly, the treatment of cells with MbetaCD concomitantly increased the phosphatidylserine externalization. Taken together, our results suggest that activation of PMCA in caveolae is modulated by phosphatidylserine, and phosphatidylserine externalization induced by MbetaCD reduced the interaction of phosphatidylserine with PMCA, subsequently PMCA-mediated Ca(2+) efflux in ECV 304 cells.     

Heparin stimulates a plasma membrane Ca2+-ATPase of Arabidopsis thaliana

We have studied the effect of heparin, a glycosaminoglycan widely used in releasing tags from fusion proteins, on isoform 8 of Arabidopsis thaliana PM Ca(2+)-ATPase (ACA8) expressed in Saccharomyces cerevisiae strain K616. Heparin stimulates hydrolytic activity of ACA8 with an estimated K(0.5) value for the complex of 15 +/- 1 microg ml(-1), which is unaffected by free [Ca(2+)]. Heparin increases V(max) up to 3-fold while it does not significantly affect the apparent K(m) for free Ca(2+) and for the nucleoside triphosphate substrate. The heparin effect is not additive with that of exogenous calmodulin and heparin is ineffective on a mutant devoid of the N-terminal auto-inhibitory domain (Delta74-ACA8). Altogether, these results indicate that heparin activation is due to partial suppression of the auto-inhibitory function of ACA8 N-terminus. Pull-down assays using heparin-agarose gel show that heparin directly interacts with ACA8. Binding to the heparin-agarose gel occurs also with a peptide reproducing ACA8 sequence (1)M-I(116). Several single-point mutations within ACA8 sequence A56-T63 significantly alter the enzyme response to heparin, suggesting that heparin interaction with this site may be involved in ACA8 activation. These results highlight a new difference between the plant PM Ca(2+)-ATPase and its animal counterpart, which is inhibited by heparin.     

The influence of membrane lipid structure on plasma membrane Ca2+ -ATPase activity

Lipid composition and Ca(2+)-ATPase activity both change with age and disease in many tissues. We explored relationships between lipid composition/structure and plasma membrane Ca(2+)-ATPase (PMCA) activity. PMCA was purified from human erythrocytes and was reconstituted into liposomes prepared from human ocular lens membrane lipids and synthetic lipids. Lens lipids were used in this study as a model for naturally ordered lipids, but the influence of lens lipids on PMCA function is especially relevant to the lens since calcium homeostasis is vital to lens clarity. Compared to fiber cell lipids, epithelial lipids exhibited an ordered to disordered phase transition temperature that was 12 degrees C lower. Reconstitution of PMCA into lipids was essential for maximal activity. PMCA activity was two to three times higher when the surrounding phosphatidylcholine molecules contained acyl chains that were ordered (stiff) compared to disordered (fluid) acyl chains. In a completely ordered lipid hydrocarbon chain environment, PMCA associates more strongly with the acidic lipid phosphatidylserine in comparison to phosphatidylcholine. PMCA associates much more strongly with phosphatidylcholine containing disordered hydrocarbon chains than ordered hydrocarbon chains. PMCA activity is influenced by membrane lipid composition and structure. The naturally high degree of lipid order in plasma membranes such as those found in the human lens may serve to support PMCA activity. The absence of PMCA activity in the cortical region of human lenses is apparently not due to a different lipid environment. Changes in lipid composition such as those observed with age or disease could potentially influence PMCA function.     

Ca2+-dependent protein kinase--a modulation of the plasma membrane Ca2+-ATPase in parotid acinar cells

Cross-talk between cAMP and [Ca(2+)](i) signaling pathways represents a general feature that defines the specificity of stimulus-response coupling in a variety of cell types including parotid acinar cells. We have reported recently that cAMP potentiates Ca(2+) release from intracellular stores, primarily because of a protein kinase A-mediated phosphorylation of type II inositol 1,4,5-trisphosphate receptors (Bruce, J. I. E., Shuttleworth, T. J. S., Giovannucci, D. R., and Yule, D. I. (2002) J. Biol. Chem. 277, 1340-1348). The aim of the present study was to evaluate the functional and molecular mechanism whereby cAMP regulates Ca(2+) clearance pathways in parotid acinar cells. Following an agonist-induced increase in [Ca(2+)](i) the rate of Ca(2+) clearance, after the removal of the stimulus, was potentiated substantially ( approximately 2-fold) by treatment with forskolin. This effect was prevented completely by inhibition of the plasma membrane Ca(2+)-ATPase (PMCA) with La(3+). PMCA activity, when isolated pharmacologically, was also potentiated ( approximately 2-fold) by forskolin. Ca(2+) uptake into the endoplasmic reticulum of streptolysin-O-permeabilized cells by sarco/endoplasmic reticulum Ca(2+)-ATPase was largely unaffected by treatment with dibutyryl cAMP. Finally, in situ phosphorylation assays demonstrated that PMCA was phosphorylated by treatment with forskolin but only in the presence of carbamylcholine (carbachol). This effect of forskolin was Ca(2+)-dependent, and protein kinase C-independent, as potentiation of PMCA activity and phosphorylation of PMCA by forskolin also occurred when [Ca(2+)](i) was elevated by the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid and was attenuated by pre-incubation with the Ca(2+) chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The present study demonstrates that elevated cAMP enhances the rate of Ca(2+) clearance because of a complex modulation of PMCA activity that involves a Ca(2+)-dependent step. Tight regulation of both Ca(2+) release and Ca(2+) efflux may represent a general feature of the mechanism whereby cAMP improves the fidelity and specificity of Ca(2+) signaling.     

Internalization of plasma membrane Ca2+-ATPase during Xenopus oocyte maturation

A transient increase in intracellular Ca²⁺ is the universal signal for egg activation at fertilization. Eggs acquire the ability to mount the specialized fertilization-specific Ca²⁺ signal during oocyte maturation. The first Ca²⁺ transient following sperm entry in vertebrate eggs has a slow rising phase followed by a sustained plateau. The molecular determinants of the sustained plateau are poorly understood. We have recently shown that a critical determinant of Ca²⁺ signaling differentiation during oocyte maturation is internalization of the plasma membrane calcium ATPase (PMCA). PMCA internalization is representative of endocytosis of several integral membrane proteins during oocyte maturation, a requisite process for early embryogenesis. Here we investigate the mechanisms regulating PMCA internalization. To track PMCA trafficking in live cells we cloned a full-length cDNA of Xenopus PMCA1, and show that GFP-tagged PMCA traffics in a similar fashion to endogenous PMCA. Functional data show that MPF activation during oocyte maturation is required for full PMCA internalization. Pharmacological and co-localization studies argue that PMCA is internalized through a lipid raft endocytic pathway. Deletion analysis reveal a requirement for the N-terminal cytoplasmic domain for efficient internalization. Together these studies define the mechanistic requirements for PMCA internalization during oocyte maturation.     

Ca2+ transport by the synaptosomal plasma membrane Ca2+-ATPase and the effect of thioridazine

Thioridazine inhibits the activity of the synaptic plasma membrane Ca(2+)-ATPase from pig brain and slightly decreases the rate of Ca(2+) accumulation by synaptic plasma membrane vesicles in the absence of phosphate. However, in the presence of phosphate, thioridazine increases the rate of Ca(2+) accumulation into synaptic plasma membrane vesicles. Phosphate anions diffuse through the membrane and form calcium phosphate crystals, reducing the free Ca(2+) concentration inside the vesicles and the rate of Ca(2+) leak. The higher levels of Ca(2+) accumulation obtained in the presence of thioridazine could be explained by a reduction of the rate of slippage on the plasma membrane ATPase.     

Ca2+-transporting properties of myometrial plasma cell membrane Ca2+, Mg2+-ATPase reconstituted in liposomes

Purified myometrium cells plasma membrane Ca2+, Mg(2+)-ATPase was reconstitute in liposomes in functionally active state by the method of cholate dialysis: it showed ATP-hydrolase activity increased by 0.8 microM A23187 average 4 times and it showed Mg2+, ATP-dependent Ca(2+)-transporting activity. Reconstituted system transported Ca2+ at an initial rate of 114.4 +/- 16.3 nmol.min-1.mg-1 with the stoichiometry Ca2+: ATP = 1: (3.2-3.7). Calmodulin increased by 30% the initial rate of Ca(2+)-accumulation by the proteoliposomes with reconstituted Ca2+, Mg(2+)-ATPase; 0.1 mM orthovanadate decreased by 80% Ca(2+)-accumulation by this system. Ca2+, Mg(2+)-ATPase reconstituted in liposomes is just Ca(2+)-transporting ATPase of the plasma membrane. Obtained enzyme preparate can be utilised for study of the properties of this important energy-dependent Ca(2+)-transporting system of smooth muscle cell.     

The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin-cholesterol domains

According to the raft hypothesis, sphingolipid-cholesterol (CHOL) microdomains are involved in numerous cellular functions. Here, we have prepared liposomes to simulate the lipid composition of rafts/caveolae using phosphatidylchone, sphingomyelin (SPM)-CHOL in vitro. Experiments of both 1,6-diphenyl-1,3,5-hexatriene and merocyanine-540 fluorescence showed that a phase transition from l(d) to l(o) can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca(2+)-ATPase (PMCA), in lipid rafts (l(o) phase). Three complementary approaches to characterize the physical appearance of PMCA were employed in the present study. Tryptophan intrinsic fluorescence increase, fluorescence quenching by both acrylamid and hypocrellin B decrease, and MIANS fluorescence decrease, indicate that the conformation of PMCA embedded in lipid l(o) phase is more compact than in lipid l(d) phase. Also, our results showed that PMCA activity decreased with the increase of SPM-CHOL content, in other words, with the increase of l(o) phase. This suggests that the specific domains containing high SPM-CHOL concentration are not a favorable place for PMCA activity. Finally, a possible explanation about PMCA molecules concentrated in caveolae/rafts was discussed.     

Evidence against involvement of the human erythrocyte plasma membrane Ca2+-ATPase in Ca2+-dependent K+ transport

Two tests were performed to assess the relationship between the Ca2+-activated K+ channel and the Ca2+-pumping ATPase in human erythrocytes. Antibodies against the purified ATPase inhibited the ATPase in resealed erythrocytes, but had no effect on the K+ channel (as assessed by Rb+ efflux). Reconstituted liposomes containing the purified active Ca2+-pumping ATPase showed no Ca2+-activated Rb+ influx. Both of these results suggest that some molecule other than the Ca2+-ATPase is responsible for the K+ channel.     

Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase

Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed.     

Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca2+-ATPase

Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca²⁺]_i) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca²⁺]_i signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca²⁺]_i, and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca²⁺]_i, which was sensitive to antioxidants and removal of external Ca²⁺, and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca²⁺-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca²⁺ uptake. This suggests that hydrogen peroxide promotes Ca²⁺ release from the endoplasmic reticulum and the mitochondria and that it promotes Ca²⁺ influx. Lower concentrations of hydrogen peroxide (10–100 µM) increased [Ca²⁺]_i and altered cholecystokinin-evoked [Ca²⁺]_i oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca²⁺]_i also upregulated the activity of the plasma membrane Ca²⁺-ATPase in a Ca²⁺-dependent manner, whereas higher concentrations (0.1–1 mM) inactivated the plasma membrane Ca²⁺-ATPase. This may be important in facilitating "Ca²⁺ overload," resulting in cell injury associated with pancreatitis. oxidant stress; pancreatitis; calcium pump     

Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium.

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.     

Reincorporated plasma membrane Ca2+-ATPase can mediate B-Type Ca2+ channels observed in native membrane of human red blood cells

Recently, we reported indirect evidence that plasma membrane Ca2+-ATPase (PMCA) can mediate B-type Ca2+ channels of cardiac myocytes. In the present study, in order to bring more direct evidence, purified PMCA from human red blood cells (RBC) was reconstituted into giant azolectin liposomes amenable to the patch-clamp technique. Purified RBC PMCA was used because it is available pure in larger quantity than cardiac PMCA. The presence of B-type Ca2+ channels was first investigated in native membranes of human RBC. They were detected and share the characteristics of cardiac myocytes. They spontaneously appeared in scarce short bursts of activity, they were activated by chlorpromazine (CPZ) with an EC50 of 149 mmole/l or 1 mmole/l vanadate, and then switched off by 10 mmole/l eosin or dose-dependently blocked by 1-5 mmole/l ATP. Independent of membrane potential, the channel gating exhibited complex patterns of many conductance levels, with three most often observed conductance levels of 22, 47 and 80 pS. The activation by vanadate suggests that these channels could play a role in the influx of extracellular Ca2+ involved in the vanadate-induced Gardos effect. In PMCA-reconstituted proteoliposomes, nearly half of the ATPase activity was retained and clear "channel-like" openings of Ba2+- or Ca2+-conducting channels were detected. Channel activity could be spontaneously present, lasting the patch lifetime or, when previously quiescent, activity could be induced by application of 50 mmole/l CPZ only in presence of 25 U/ml calmodulin (CaM), or by application of 1 mmole/l vanadate alone. Eosin (10 mmole/l) and ATP (5 mmole/l) significantly reduced spontaneous activity. Channel gating characteristics were similar to those of RBC, with main conductance levels of 21, 40 and 72 pS. The lack of direct activation by CPZ alone might be attributed to a purification-induced modification or absence of unidentified regulatory component(s) of PMCA. Despite a few differences in results between RBC and reincorporated PMCA, most probably attributable to the decrease in ATPase activity following the procedure of reincorporation, the present experimental conditions appear to reveal a channel-mode of the PMCA that shares many similarities with the B-type Ca2+ channel.     

The interaction of ethanol with reconstituted synaptosomal plasma membrane Ca2+ -ATPase

The primary effect of ethanol is on the central nervous system. However, the molecular mechanisms responsible for the physiological symptoms of ethanol intoxication are still unknown. Low concentrations of ethanol were observed to stimulate the activity of the calcium pump from reconstituted synaptosomal plasma membrane Ca2+ -ATPase (PMCA), and ethanol inhibited Ca2+ -ATPase activity at concentrations above 5%. The greatest stimulating effect was obtained with 5% (v/v) ethanol and was lipid-dependent, being 74% when the protein had been reconstituted in phosphatidylcholine (PC) and less when the reconstituted protein had previously been activated by calmodulin or after removal of a 9-kDa autoinhibitory site by controlled trypsinization. Stimulation of the pump by ethanol was lower for the native or trypsin-digested protein in the presence of phosphatidylserine than in PC. These results suggest a direct ethanol-protein interaction, because the activating effect depended on the state of Ca2+ -ATPase (native or truncated, or in presence of calmodulin). The activating mechanism of ethanol may involve opening an autoinhibitory domain located close to the calmodulin binding domain.     

A high-affinity plasma membrane Ca2+-ATPase in Dictyostelium discoideum: its relation to cAMP-induced Ca2+ fluxes

Chemotactic stimulation of Dictyostelium discoideum induces an uptake of Ca2+ by the cells followed by a release of Ca2+. In this study we investigated the mechanism of Ca2+ release and found that it was inhibited by La3+, Cd2+ and azide. Ca2+ release occurred in the absence of external Na+, indicating that an Na+/Ca2+ exchange was not involved. Plasma membranes contained high- and low-affinity ATPase activities. Apparent K0.5 values were 8 microM for the major Mg2+-ATPase and 1.1 microM for the high-affinity Ca2+-ATPase, respectively. The Mg2+-ATPase activity was inhibited by elevated concentrations of Ca2+, whereas both Ca2+-ATPases were active in the absence of added Mg2+. The activities of the Ca2+-ATPases were not modified by calmodulin. The high-affinity Ca2+-ATPase was competitively inhibited by La3+ and Cd2+; we suggest that this high-affinity enzyme mediates the release of Ca2+ from D. discoideum cells.     

Stimulation of plant plasma membrane Ca2+-ATPase activity by acidic phospholipids

The effect of phospholipids on the activity of the plasma membrane (PM) Ca²⁺-ATPase was evaluated in PM isolated from germinating radish ( Raphanus sativus L. cv. Tondo Rosso Quarantino) seeds after removal of endogenous calmodulin (CaM) by washing the PM vesicles with EDTA. Acidic phospholipids stimulated the basal Ca²⁺-ATPase activity in the following order of efficiency: phosphatidylinositol 4,5-diphosphate (PIP2)≈phosphatidylinositol 4-monophosphate>phosphatidylinositol≈phosphatidylserine≈phosphatidic acid. Neutral phospholipids as phosphatidylcholine and phosphatidylethanolamine were essentially ineffective. When the assays were performed in the presence of optimal free Ca²⁺ concentrations (10 μ M ) acidic phospholipids did not affect the Ca²⁺-ATPase activated by CaM or by a controlled trypsin treatment of the PM, which cleaved the CaM-binding domain of the enzyme. Analysis of the dependence of Ca²⁺-ATPase activity on free Ca²⁺ concentration showed that acidic phospholipids increased V_max and lowered the apparent K_m for free Ca²⁺ below the value measured upon tryptic cleavage of the CaM-binding domain; in particular, PIP2 was shown to lower the apparent K_m for free Ca²⁺ of the Ca²⁺-ATPase also in trypsin-treated PM. These results indicate that acidic phospholipids activate the plant PM Ca²⁺-ATPase through a mechanism only partially overlapping that of CaM, and thus involving a phospholipid-binding site in the Ca²⁺-ATPase distinct from the CaM-binding domain. The physiological implications of these results are discussed.     

Calcium uptake and Ca2+-ATPase activity in goat spermatozoa membrane vesicles do not require Mg2+

Microsomal membrane vesicles isolated from goat spermatozoa contain Ca²⁺-ATPase, and exhibit Ca²⁺ transport activities that do not require exogenous Mg²⁺ .The enzyme activity is inhibited by calcium-channel inhibitors,e.g. verapamil and diltiazem, like the well known Ca²⁺ , Mg²⁺-ATPase. The uptake of calcium is ATP (energy)-dependent and the accumulated Ca²⁺ can be completely released by the Ca²⁺ ionophore A23187, suggesting that a significant fraction of the vesicles are oriented inside out