Enhancement of plasma membrane (Ca2+-Mg2+)-ATPase activity in regenerating rat liver: Involvement of endogenous activating protein regucalcin

The alteration of (Ca²⁺-Mg²⁺)-ATPase activity in the plasma membranes of regenerating rat liver after a partial hepatectomy was investigated. Liver was surgically removed about two thirds of that of sham-operated rats. The reduced liver weight by partial hepatectomy was restored about 50% at 24 h after the surgery, and it was completely restored at 72 h. Regenerating liver significantly increased calcium content and plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity between 12–48 h after hepatectomy. Those increases were maximum at 24 h after the surgery. The regenerating liver-induced increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was completely abolished by the presence of anti-regucalcin IgG (1.0–4.0 g/ml). The regenerating liver-induced increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was clearly inhibited by N-ethylmaleimide (2.5 and 5.0 mM) addition into the enzyme reaction mixture. This NEM effect was also seen for the activatory effect with regucalcin (0.25 M) addition on the enzyme activity in the plasma membranes from normal rat liver. The endogenous regucalcin may play a cell physiological role in the activation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase to maintain the intracellular calcium level in regenerating rat liver.     

Activating effect of regucalcin on (Ca2+-Mg2+)-ATPase in rat liver plasma membranes: relation to sulfhydryl group

The activating mechanism of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase in the plasma membranes of rat liver was investigated. (Ca²⁺–Mg²⁺)-ATPase activity was markedly increased by a sulfhydryl (SH) group protecting reagent dithiothreitol (DTT; 2.5 and 5 mM as a final concentration), while the enzyme activity was significantly decreased by a SH group modifying reagent N-ethylmaleimide (NEM; 0.5–5 mM). The effect of DTT (5 mM) to increase the enzyme activity was clearly blocked by NEM (5 mM). Regucalcin (0.25–1.0 M) significantly increased (Ca²⁺-Mg²⁺)-ATPase activity. This increase was completely blocked by NEM (5 mM). Meanwhile, digitonin (0.04%), which can solubilize the membranous lipids, significantly decreased (Ca²⁺–Mg²⁺)-ATPase activity. Digitonin did not have an effect on the DTT (5 mM)-increased enzyme activity. However, the effect of regucalcin (0.25 M) increasing (Ca²⁺–Mg²⁺)-ATPase activity was entirely blocked by the presence of digitonin. The present results suggest that regucalcin activates (Ca²⁺–Mg²⁺)-ATPase by the binding to liver plasma membrane lipids, and that the activation is involved in the SH groups which are an active site of the enzyme.     

Streptozotocin-induced diabetes increases (Ca2+-Mg2+)-ATPase activity in hepatic plasma membranes of rats: Involvement of protein kinase C

The alteration in calcium transport in the liver of rats with streptozocin(STZ)-diabetic state was investigated. STZ (6 mg/100 g body weight) was subcutaneously administered in rats, and 1 or 2 weeks later they were sacrificed by bleeding. STZ administration caused a remarkable elevation of serum glucose concentration. Liver calcium content was significantly increased by STZ administration. Hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was markedly elevated by STZ administration. This increase was completely abolished by the presence of staurosporine (10^-7-10^-5 M), an inhibitor of protein kinase C, in the enzyme reaction mixture, suggesting an involvement of protein kinase C signalling. Moreover, the STZ-induced increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was significantly raised by the presence of okadaic acid (10^-5 and 10^-4 M). Meanwhile, the STZ-increased (Ca²⁺-Mg²⁺)-ATPase activity was not appreciably altered by the presence of anti-regucalcin IgG in the reaction mixture, indicating that the activatory protein regucalcin does not participate in the elevation of the enzyme activity. The present study demonstrates that STZ-induced diabetes causes the increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity of rats.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

Human thymus poly(A) polymerase (EC 2.7.7.19) activity has been investigated using poly(A) and oligo(A) as initiators. All obtained fractions reveal more than one polypeptide as detected by immunoblotting after SDS-PAGE. In addition to the homogeneously purified (Tsiapalis et al., J Biol Chem 250: 4486–1496, 1975 and Wahle, J Biol Chem 266: 3131–3139, 1991), about 60 kDa polypeptide, a larger polypeptide, about 80 kDa, that comigrates in the region of poly(A) polymerase activity was detected, enriched and partially characterized; it appears having similar size with bovine poly(A) polymerase cloned in E. coli. Polyclonal antiserum produced against recombinant bovine poly(A) polymerase reacts more efficiently with the about 80 kDa polypeptide upon immunoblotting, and can precipitate the poly(A) polymerase activity. This enzyme form, from human tissue, is novel in terms of size and may reflect intact or physiological form of poly(A) polymerase in human thymus, and supports and substantiates recent reports on the enzyme from other sources.     

Stimulatory effect of regucalcin on ATP-dependent calcium transport in rat liver plasma membranes

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytoplasm, on ATP-dependent calcium transport in the plasma membrane vesicles of rat liver was investigated. (Ca2+-Mg2+)-ATPase activity in the liver plasma membranes was significantly increased by the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the enzyme reaction mixture. This increase was completely inhibited by the presence of sulfhydryl group modifying reagent Nethylmaleimide (5.0 mM NEM) or digitonin (0.04%), which can solubilize the membranous lipids. When ATP-dependent calcium uptake by liver plasma membrane vesicles was measured by using 45CaCl2, the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the reaction mixture caused a significant increase in the 45Ca2+ uptake. This increase was about 2-fold with 0.5 \sgmaelig;M regucalcin addition. An appreciable increase was seen by 5 min incubation with regucalcin addition. The regucalcin-enhanced ATP-dependent 45Ca2+ uptake by the plasma membrane vesicles was completely inhibited by the presence of NEM (5.0 mM) or digitonin (0.04%). These results demonstrate that regucalcin activates (Ca2+-Mg2+)-ATPase in the liver plasma membranes and that it can stimulate ATP-dependent calcium transport across the plasma membranes.     

Activatory effect of regucalcin on GTPase activity in rat liver plasma membranes

The effect of regucalcin, a regulatory protein of Ca²⁺ signaling, on guanosine-5-triphosphatase (GTPase) activity in isolated rat liver plasma membranes was investigated. GTPase activity was significantly increased by the addition of Ca²⁺ (25–100 M) in the enzyme reaction mixture. Such an increase was not seen by other metals (Mg, Co, Zn, Cu, Ni, and Mn) with 50 M. The activatory effect of calcium (50 M) was significantly decreased by calmodulin (2.5 and 5 g/ml), indicating that it does not depend on calmodulin. The presence of regucalcin (0.1–0.5 M) in the enzyme reaction mixture caused a significant increase in GTPase activity. This increase was not significantly enhanced by calcium (50 M). GTPase activity was significantly increased by dithiothreitol (DTT; 5 mM), a protecting reagent of thiol (SH)-groups, while it was decreased by N-ethylmaleimide (NEM; 5 mM), a modifying reagent of SH-groups. The effect of calcium or regucalcin in increasing GTPase activity was not seen in the presence of NEM. Also, the activatory effect of calcium or regucalcin on GTPase was not seen in the presence of vanadate, an inhibitor of protein phosphorylation, which could inhibit GTPase activity. Moreover, the effect of regucalcin was not seen in the presence of digitonin (0.01%), a solubilizing reagent of membranous lipids, while the effect of calcium was not inhibited by digitonin. The present study demonstrates that regucalcin has an activatory effect on GTPase activity independently of Ca²⁺ in rat liver plasma membranes.     

p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+)

The plasma membrane (Ca²⁺+Mg²⁺)ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca²⁺ ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca²⁺ strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca²⁺-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca²⁺ is related to opposite long-term hydration effects on the substrate binding domain and the Ca²⁺ binding domain.     

The thermodynamic efficiency of the Ca2+-Mg2+-ATPase is one hundred percent

The thermodynamic efficiency of the Ca²⁺-Mg²⁺-ATPase of skeletal sarcoplasmic reticulum has been evaluated by comparing the Ca²⁺ gradient established with the ATP/(ADP*P_i) ratio. The evaluation was made at an external Ca²⁺ level (4.7 × 10^–8 M) which is below theK _m value of 7 × 10^–8 M. The Mg-ATP and phosphate concentrations were held constant (0.1 mM) and the ADP concentration was varied. Maximal uptake to an internal free Ca²⁺ concentration of 17 mM was observed at infinite ATP/(ADP*P_i) ratio (absence of ADP). This corresponds to a [Ca²⁺]_i/[Ca²⁺]₀ gradient of 3.6 × 10⁵. A Ca²⁺ gradient one-half as large was observed at an ATP/(ADP*P_i) ratio of 3.5 × 10³ M^–1. The square of the Ca²⁺ gradient is shown to be proportional to the ATP/(ADP*P_i) ratio, for finite values of the latter. The proportionality constant is identical to the equilibrium constant for hydrolysis of ATP (9.02 × 10⁶ M) under these conditions (0.1 mM Mg²⁺, 30°C). The intrinsic thermodynamic efficiency of the pump is shown to be 100%, with a maximal uncertainty of 3%. The efficiency is lower under less optimal conditions, when the pump is inhibited and passive leak processes compete.Dedicated to Prof. Philip George, University of Pennsylvania, whose instruction, research, and example made this contribution possible.     

Enhancement of nuclear Ca2+-ATPase activity in regenerating rat liver: involvement of nuclear DNA increase

The alteration of calcium content, Ca²⁺-ATPase activity, DNA content and DNA fragmentation in the nuclei of regenerating rat liver was investigated. Liver was surgically removed about 70% of that of sham-operated rats. the reduced liver weight by partial hepatectomy was completely restored at 3 days after the surgery. Regenerating liver significantly increased Ca²⁺-ATPase activity and DNA content in the nuclei between 1 and 5 days after hepatectomy. The nuclear calcium content was clearly increased from 2 days after hepatectomy. The increase of Ca²⁺-ATPase activity in regenerating liver was clearly inhibited by the presence of trifluoperazine (10 M), staurosporine (2.5 M) and dibucaine (10 M), which are inhibitors of calmodulin and protein kinase, in the enzyme reaction mixture. However, the nuclear enzyme activity in normal rat liver was not significantly altered by these inhibitors. Meanwhile, the increase of nuclear DNA content in regenerating liver was completely blocked by the administration of trifluoperazine (2.5 mg/100 g body weight), suggesting an involvement of calmodulin. Now, the nuclear DNA fragmentation was significantly decreased in regenerating liver, suggesting that this decrease is partly contributed to the increase in nuclear DNA content. The present study clearly demonstrates that regenerating liver enhances nuclear Ca²⁺-ATPase activity and induces a corresponding elevation of nuclear calcium content. This Ca²⁺-signaling system may be involved in the regulation of nuclear DNA functions in regenerating rat liver.     

Regulation of cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary The two high affinity calcium binding sites of the cardiac (Ca²⁺ + Mg²⁺)-ATPase have been identified with the use of Eu³⁺. Eu³⁺ competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of H₂O molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca²⁺ sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6–8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites.     

Purification of the microsomal Ca2+-ATPase from rat liver

Summary The Ca²⁺-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficollsucrose treatment, column chromatography with agarose-hexane adenosine 5-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca²⁺-ATPase was stable for at least two weeks when stored at –70°C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca²⁺-ATPase. Further characterization of the ER Ca²⁺-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca²⁺-ATPase cross-reacted with the purified Ca²⁺-ATPase from rat liver ER membranes.     

Neutral organic solute effects on the activity of the plasma membrane Ca2+-ATPase

We have compared effects of dimethylsulfoxide (Me₂SO) and two polyols on the Ca²⁺-ATPase purified from human erythrocytes. As studied under steady-state conditions over a broad solute concentration range and temperature, Me₂SO, glycerol, and xylitol do not inhibit the Ca²⁺-ATPase activity; this is in contrast to numerous other organic solutes that we have investigated. Under specific experimental conditions, Me₂SO (but not glycerol) substantially increases Ca²⁺-ATPase activity, suggesting a possible facilitation of enzyme oligomerization. The activation is more pronounced at low Ca²⁺ concentrations. In contrast to glycerol, Me₂SO shows no protective effect on enzyme structure as assessed by determining residual Ca²⁺-ATPase activity after exposing the enzyme to thermal denaturation at 45°C. Under these conditions several other organic solutes strongly enhance the denaturating effect of temperature. Because of the temperature dependence of its effect on the Ca²⁺-ATPase activity we believe that Me₂SO activates the Ca²⁺-ATPase by indirect water-mediated interactions.     

Erythrocyte membrane (Ca2++Mg2+)-ATPase in human protein-energy malnutrition

Calmodulin-free ghost membranes were prepared from erythrocytes of kwashiorkor children and from healthy children in the same age bracket. In the absence of calmodulin, the specific activity of Mg²⁺-dependent Ca²⁺-pumping ATPase (Ca²⁺+Mg²⁺-ATPase) of kwashiorkor membranes was more than 40 percent lower than the specific activity of the normal enzymes, whose maximum velocity was increased by at least four-fold by the modulator protein. In constrast, the maximum velocity of the enzymes of kwashiorkor membranes was enhanced by calmodulin by about 11/2 times the basal activity of the normal enzymes and by 2 times the basal activity of the kwashiorkor enzymes. The affinity of the pump for ATP was lower in the membranes of kwashiorkor children (K_m for ATP=30.6±2.8 M ATP) in comparison to normal membranes (K_m for ATP=21.7±2.0 M ATP). Similarly, calmodulin-affinity of the enzymes, was lower in kwashiorkor membranes than in the normal membranes irrespective of source of calmodulin. Calmodulin from haemolysates of kwashiorkor red cells activated the enzymes of normal and kwashiorkor membranes to the same degree as calmodulin partially purified from the haemolysate of healthy children. A determination of the dependence of the activity of the pump on calcium in the absence and presence of calmodulin reveals that the affinity of the kwashiorkor enzymes for Ca²⁺ is at least 70 percent lower than that of enzymes of normal membranes. Altogether, these findings suggest that the Ca²⁺-pumping ATPase of kwashiorkor membranes is less functional than the enzymes of healthy erythrocytes.     

Potential role of regucalcin as a specific biochemical marker of chronic liver injury with carbon tetrachloride administration in rats

The potential sensitivity of liver specific protein regucalcin as a biochemical marker of chronic liver injury with carbon tetrachloride (CCl₄) administration in rats was investigated. CCl₄ (10%; 1.0 ml/100 g body wt) was orally given 5 times at 3-day intervals to rats, and the animals were killed by bleeding at 3, 6, 18, and 30 days after the first administration of CCl₄. The body weight of rats was significantly lowered 3 and 6 days after CCl₄ administration as compared with that of control rats administered with corn oil, and then the weight was restored at 18 and 30 days. Serum glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities were significantly increased 3 days after the administration, while a significant increase in serum -glutamyltranspeptidase (-GTP) activity was seen at 3 and 6 days after the administration. Serum GOT, GPT, and -GTP activities were restored to control levels at 18 and 30 days after CCl₄ administration. Serum albumin, -fetoprotein, and ammonium levels were not changed by CCl₄ administration. Meanwhile, serum regucalcin concentration was markedly increased 3 and 6 days after CCl₄ administration, and a significant increase in serum regucalcin concentration was observed 18 and 30 days after the administration. Liver regucalcin mRNA and liver cytosolic regucalcin levels were significantly decreased 18 and 30 days after CCl₄ administration. Liver content of calcium, which intracellular calcium homeostasis is maintained, was significantly increased between 3 and 30 days after CCl₄ administration. Hepatic mitochondrial succinate dehydrogenase activity was significantly increased 30 days after the administration. The present study demonstrates that serum regucalcin has a potential sensitivity as a specific biochemical marker of chronic liver injury with CCl₄ administration in rats.     

Disruption of energy transduction in sarcoplasmic reticulum by trypsin cleavage of (Ca2++Mg2+)-ATPase

Summary Proteolytic digestion of sarcoplasmic reticulum vesicles with trypsin has been used as a structural modification with which to examine the interaction between the ATP hydrolysis site and calcium transport sites of the (Ca²⁺+Mg²⁺)-ATPase. The kinetics of trypsin fragmentation were examined and the time course of fragment production compared with ATP hydrolytic and calcium uptake activities of the digested vesicles. The initial cleavage (TD 1) of the native ATPase to A and B peptides has no effect on the functional integrity of the enzyme, hydrolytic and transport activities remaining at the levels of the undigested control. Concomitant with the second tryptic cleavage (TD 2) of the A peptide to A₁ and A₂ fragments, calcium transport is inhibited. Kinetic analysis demonstrates that the rate constant for inhibition of calcium uptake is correlated with the rate constant of a fragment disappearance. Both Ca²⁺-dependent and total ATPase activities are unaffected by this second cleavage. Passive loading of vesicles with calcium and subsequent efflux measurements show that transport inhibition is not due to increased permeability of the membrane to calcium even at substantial extents of digestion. Steady-state levels of acidstable phosphoenzyme are unaffected by either TD 1 or TD 2, indicating that uncoupling of the hydrolytic and transport functions does not increase the turnover rate of the enzyme and that TD 2 does not change the essential characteristics of the ATP hydrolysis site. Sarcoplasmic reticulum (SR) vesicles were examined for the presence of tightly bound nucleotides and are shown to contain 2.8–3.0 nmol ATP and 2.6–2.7 nmol ADP per mg SR protein. The ADP content of SR remains essentially unchanged with TD 1 cleavage of the ATPase enzyme to A and B peptides, but declines upon TD 2 in parallel with the digestion of the A fragment and the loss of calcium uptake activity of the vesicles. The ATP content is essentially constant throughout the course of trypsin digestion. The results are discussed in terms of current models of the SR calcium pump and the molecular mechanism of energy transduction.     

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.     

Uncoupling of Occlusion From ATP Hydrolysis Activity in Sarcoplasmic Reticulum (Ca2++Mg2+)-ATPase

The uncoupling of Ca²⁺ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu³⁺ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Co²⁺ and at 25°C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca²⁺, 3 mM ATP, and at 25°C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu³⁺. Digest past TD2 in the presence of 5 mM Ca²⁺ and 3 mM AMP-PNP. (a non-hydrolyzable ATP analog) at 25°C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca²⁺ transport from ATPase activity is possible by tryptic digestion of the (Ca²⁺ + Mg²⁺)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.     

Effect of nuclear Ca2+ uptake inhibitors on Ca2+-activated DNA fragmentation in rat liver nuclei

The effect of nuclear Ca²⁺ uptake inhibitors on the Ca²⁺-activated DNA fragmentation in rat liver nuclei was investigated. The addition of Ca²⁺ (40 M) into the reaction mixture containing liver nuclei in the presence of 2.0 mM ATP caused a remarkable increase in nuclear DNA fragmentation. This Ca²⁺-activated DNA fragmentation was not seen in the absence of ATP, because nuclear Ca²⁺ uptake is not initiated without ATP addition. Moreover, the presence of various reagents (10 M arachidonic acid, 2.0 mM NAD⁺, 10 M zinc sulfate and 0.2 mM N-ethylmaleimide), which could inhibit Ca²⁺-ATPase activity and Ca²⁺ uptake in the nuclei, produced a significant inhibition of the Ca²⁺-activated DNA fragmentation in the nuclei. The results show that the Ca²⁺-activated DNA fragmentation is involved in the uptake of Ca²⁺ by the nuclei, suggesting a role of Ca²⁺ transport system in the regulation of liver nuclear functions.     

The plasma membrane (Mg2+)-dependent adenosine triphosphatase from the human erythrocyte is not an ion pump

Summary The plasma membrane (Mg²⁺)-dependent adenosine triphosphatase ((Mg²⁺)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg²⁺)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H⁺ transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca²⁺)-dependent adenosine triphosphatase ((Ca²⁺)-ATPase) activity. Proton efflux associated with (Ca²⁺)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca²⁺)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982)J. Biol. Chem. 257:2350–2356)) and was shown to be stimulated by calmodulin. The ability of the (Mg²⁺)-ATPase to pump²⁸Mg²⁺,³⁵SO ₄ ^2– and⁸⁶Rb⁺ was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system.