Incorporation of Na+ - Ca2+ antiporter and of (Na+ + K+)-ATPase into liposomes and demonstration of their non-identity

(Na+ + K+)-ATPase was isolated from the grey matter of brain and incorporated into liposomes. Most of the reconstituted enzyme was oriented 'inside-out' with respect to its in vivo orientation and externally added ATP promoted Na+ uptake that was inhibitable by internally trapped ouabain. Using the same proteoliposomes, an Na+ - Ca2+ exchange system was observed as indicated by the following pieces of evidence. (1) The Na+ gradient provided the only readily apparent driving force for acceleration of Ca2+ accumulation into proteoliposomes. (2) The antiporter was specific for Ca2+, high Mg2+ excess did not inhibit Ca2+ antiport. (3) The Na+ efflux was dependent on the extravesicular Ca2+ concentration. (4) The Na+ efflux was not inhibited by tetrodotoxin. The demonstrated Na+ - Ca2+ exchange could not be related to (Na+ + K+)-ATPase protein, since it was not purified with (Na+ + K+)-ATPase, as followed from transport studies with liposomes containing (Na+ + K+)-ATPase of different specific activity. The results strongly indicate that plasma membranes isolated from the grey matter of brain contain an Na+ - Ca2+ exchange system and that the proteoliposomes are suitable for further purification of the carrier molecule.     

The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membranes is a Ca2+ pump. Reconstitution of the purified enzyme into phospholipid vesicles

The purified (Ca2+-Mg2+)-ATPase from rat liver plasma membranes (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215) was incorporated into soybean phospholipid vesicles, together with its activator. In the presence of millimolar concentrations of Mg2+, the reconstituted proteoliposomes displayed a rapid, saturable, ATP-dependent Ca2+ uptake. Half-maximal Ca2+ uptake activity was observed at 13 +/- 3 nM free Ca2+, and the apparent Km for ATP was 16 +/- 6 microM. Ca2+ accumulated into proteoliposomes (2.8 +/- 0.2 nmol of Ca2+/mg of protein/90 s) was totally released upon addition of the Ca2+ ionophore A-23187. Ca2+ uptake into vesicles reconstituted with enzyme alone was stimulated 2-2.5-fold by the (Ca2+-Mg2+)-ATPase activator, added exogenously. The (Ca2+-Mg2+)-ATPase activity of the reconstituted vesicles, measured using the same assay conditions as for ATP-dependent Ca2+ uptake activity (e.g. in the presence of millimolar concentrations of Mg2+), was maximally activated by 20 nM free Ca2+, half-maximal activation occurring at 13 nM free Ca2+. The stoichiometry of Ca2+ transport versus ATP hydrolysis approximated 0.3. These results provide a direct demonstration that the high affinity (Ca2+-Mg2+)-ATPase identified in liver plasma membranes is responsible for Ca2+ transport.     

The blocking effect of aliphatic hydrocarbons on Ca2+ leakage channels formed by aggregates of Ca2+-ATPase of the sarcoplasmic reticulum

The effects of aliphatic hydrocarbons within the liposomes on the Ca2+ transport function of isolated sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle, vesiculate preparation of Ca2+ dependent ATPase and proteoliposomes reconstituted from Ca2+-ATPase and egg phosphatidylcholine, were studied. It was shown that liposomes prepared from dipalmitoyl phosphatidylcholine containing aliphatic hydrocarbons increase 2 to 3 times Ca2+ accumulation by Ca2+-dependent ATPase from rabbit skeletal muscle SR. Ca2+ transport by SR vesicles increases in the presence of hydrocarbons by 15--20%. The activating effect of hydrocarbons on Ca2+ transport by proteoliposomes depends on the lipid/protein ratio. The proteoliposomes with a high lipid/protein ratio are practically insensitive to the effects of hydrocarbons. It was suggested that activation of Ca2+ transport by hydrocarbons is due to blocking of Ca2+ leakage channels formed during the aggregation of Ca2+-ATPase molecules. Treatment of membranes by formaldehyde results in the oligomerization of Ca2+-ATPase and decreases 2--4-fold the ATP-dependent accumulation of Ca2+. Subsequent addition of decane restores Ca2+ transport practically completely.     

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.     

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.     

Inhibition of red cell Ca2+-ATPase by vanadate

1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate. 2. Several natural activators of Ca2+-ATPase (Mg2+, K+, Na+ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate. 3. Among the ligands tests, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K 1/2 for vanadate was 1.5 microM and inhibition was nearly complete at saturating vanadate concentrations. 5. There are similarities between the kinetics of inhibition of red cell Ca2+-ATPase and (Na+ + K+)-ATPase prepared from a variety of sources; however, (Na+ + K+)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.     

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+.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.     

Chemical modification of sarcoplasmic reticulum with methylbenzimidate. Stimulation of Ca2+ efflux.

Treatment of sarcoplasmic reticulum membranes with 12 mM-methylbenzimidate (MBI) for 5 min, in the presence of 5 mM-ATP at pH 8.5, resulted in a 2-3-fold stimulation of ATP hydrolysis and over 90% inhibition of Ca2+ accumulation. This phenomenon was strictly dependent upon the presence of nucleotides with the following order of effectiveness: adenosine 5'-[beta, gamma-imido]triphosphate greater than or equal to ATP greater than UTP greater than ADP greater than AMP. Divalent cations such as Ca2+, Mg2+ and Mn2+, when present during the MBI treatment, prevented both the stimulation of ATPase activity and the inhibition of Ca2+ accumulation. Modification with MBI had no effect on E-P formation from ATP, ADP-ATP exchange, Ca2+ binding or ATP-Pi exchange catalysed by the membranes. Membranes modified with MBI in the presence of ATP and then passively loaded with Ca2+ released about 80% of their Ca2+ content within 3 s. Control membranes released only 3% of their Ca2+ during the same time period. MBI modification inhibited Ca2+ accumulation by proteoliposomes reconstituted with the partially purified ATPase but not with the purified ATPase fraction. These results suggest that MBI in the presence of ATP stimulates Ca2+ release by modifying a protein factor(s) other than the (Ca2+ + Mg2+)-ATPase.     

Changes in microsomal Na+, K+-, Mg2+- and Ca2+-ATPase activities during proliferation of Chinese hamster V-79 and human HeLaS-3 cells

Changes in microsomal Na+, K+-, Mg2+- and Ca2+-ATPase activities during cell proliferation were examined in Chinese hamster V-79 (V-79) cells (normal cells) and human HeLaS-3 (HeLaS-3) cells (malignant cells). For V-79 cells, the Mg2+-ATPase activity per cell (pmol Pi/h/cell) in the confluent phase was higher than that in the logarithmically growing (log) phase. The amount of microsomal protein per cell was also high in the confluent phase. Specific activities (mumol Pi/h/mg protein) of Na+, K+-, Mg2+- and Ca2+-ATPase were significantly lower in the confluent phase than in the log phase. For HeLaS-3 cells, an increase in Ca2+-ATPase activity per cell was observed. The amount of microsomal protein per cell did not change between the log and confluent phase. The specific activity of Ca2+-ATPase in the confluent phase was also markedly higher than in the log phase. The relation between changes in ATPase activities and cell proliferation is discussed.     

Fe2+ and other divalent metal ions uncouple Ca2+ transport from (Ca2+-Mg2+)-ATPase in rat liver plasma membranes

The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.     

Effect of gradients of monovalent cations on active transport of Ca2+ in the sarcoplasmic reticulum and proteoliposomes

Artificially generated K+ gradient from the sarcoplasmic reticulum vesicles enhances the ATP-dependent Ca2+ transport. The effect is not specific for K+, and is observed when K+ is replaced by Na+ or choline. Dissipation of the K+, Na+, choline gradient does not influence the ATP-dependent Ca2+ transport in proteoliposomes from asolectin and purified Ca2+-ATPase. The K gradient in the presence of valinomycin stimulates the ATP-dependent Ca2+ transport in proteoliposomes.     

Distribution of Ca2+-ATPase, ATP-dependent Ca2+-transport, calmodulin and vitamin D-dependent Ca2+-binding protein along the villus-crypt axis in rat duodenum

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca2+-ATPase activity and ATP-dependent Ca2+-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na+ + K+)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca2+-transport in basolateral membranes was highest in fraction II (8.2 +/- 0.3 nmol Ca2+/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca2+-transport activity (0.9 +/- 0.1 nmol Ca2+/min per mg protein). The distribution of high-affinity Ca2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca2+-transport. The activity of Na+/Ca2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca2+-transport system, the Na+/Ca2+ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.     

Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

The plasma membrane Ca2+-ATPase protein from red blood cells is not modified in preeclampsia

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.     

Investigation of (Ca2+ + Mg2+)-ATPase phosphoprotein formation in erythrocyte membranes of patients with cystic fibrosis

The (Ca2+ + Mg2+)-ATPase present per mg of protein in erythrocyte membranes of controls and patients with cystic fibrosis (CF) was determined by estimation of the levels of its phosphoprotein. In the presence of 10 mM free Ca2+, which inhibits phosphoprotein decomposition, significantly less phosphoprotein intermediate, ECaP, was found in erythrocyte membranes from CF patients than in age- and sex-matched controls; this correlated with a significant decrease in (Ca2+ + Mg2+)-ATPase activity. These observations indicate a decrease in the number of functional (Ca2+ + Mg2+)-ATPase molecules in erythrocyte membranes from CF patients or an alteration in either the structure of the pump protein or the composition of its environment.     

Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells

Ca2+-ATPase was isolated from plasma membranes of Ehrlich ascites mammary carcinoma cells by means of calmodulin affinity chromatography. The purification procedure included removal of endogenous calmodulin from a Triton X-100 solubilizate of the membranes by DEAE ion-exchange chromatography as an essential step. With respect to its molecular mass, activation by calmodulin, Ca2+-dependent phosphorylation and highly sensitive inhibition by orthovanadate, the purified enzyme resembles the Ca2+-ATPase of erythrocyte membranes. In contrast to the strong calmodulin dependence of the isolated enzyme the Ca2+-ATPase in native Ehrlich ascites carcinoma cell membranes cannot be remarkably stimulated by added calmodulin. It is suggested that the membrane-bound Ca2+-ATPase in the presence of Ca2+ is activated by interaction with endogenously bound calmodulin.     

Activation of erythrocyte membrane Ca2+-ATPase by calpain

Ca2+-ATPase of erythrocyte membranes, prepared from erythrocytes substantially removed of contaminating leukocytes, was found to be activated by calpain isolated from the same source. Saponin or glycodeoxycholate treatment of membranes was essential for elicitation of the calpain response. Unlike the membrane bound ATPase, solubilized ATPase was inactivated by calpain. Digestion of membranes with the protease did not affect the Km (ATP) of Ca2+-ATPase though stimulation of the membrane ATPase by calmodulin could be partially substituted by calpain treatment. As compared with control, Ca2+-ATPase of calpain-digested membranes attained maximal activity at a lower free Ca2+ concentration.     

The role of Ca2+-ATpase and its hydrophobic component in the release of Ca2+ from skeletal muscle sarcoplasmic reticulum

The Ca2+ permeability of proteoliposomes containing Ca2+-ATPase of sarcoplasmic reticulum and its hydrophobic fragment was investigated, using the method of synthetic penetrant ions and the radioisotopic method. The former method was used to determine the diffusional membrane potential formed by Ca2+ concentration gradient. It was demonstrated that Ca2+-ATPase, whose active center is oriented outside, has and asymmetric conductivity, i. e., it facilitates the rapid efflux of Ca2+ from proteoliposomes. This efflux is stimulated by the membrane potential positive inside. The hydrophobic fragment of Ca2+-ATPase forms a Ca2+-channel with a high conductivity for Ca2+. This channel is responsible for the Ca2+ efflux from sarcoplasmic reticulum.