Effects of pH, temperature, and calcium concentration on the stoichiometry of the calcium pump of sarcoplasmic reticulum

Coupling of Ca2+ transport to ATP hydrolysis by isolated skeletal muscle sarcoplasmic reticulum vesicles has been investigated by means of ATP pulse methods. The stoichiometric amounts of Ca2+ transported per pulse of ATP were measured by Ca2+-stat methods, using either a Ca2+ electrode or arsenazo III as end point detectors, or by means of 45CaCl2. Maximum coupling ratios (Ca2+/ATP), of 1.82 +/- 0.13 occurred at pH 6.8, 25 degrees C, and in the presence of saturating Ca2+ concentrations. Ca2+/ATP values decreased at alkaline pH, with an apparent pK alpha of 7.9. The coupling ratio was unaltered between 6 and 30 degrees C, but decreased to 0.4 at 42 degrees C. Uncoupling by alkaline pH and high temperatures was reversible. The coupling process was Ca2+-dependent, with a K0.5 value for Ca2+ of 0.12 microM and a Hill coefficient of 2.0. Ca2+ ions, which were transported into vesicles under conditions resulting in low coupling ratios, were retained as the calcium oxalate precipitate, following complete hydrolysis of substrate. Passive Ca2+ efflux and Ca2+ exchange, were independent of pH. The observed variations in Ca2+/ATP ratio cannot readily be explained on the basis of a pump-leak model. Rather, the Ca2+-ATPase appears to be capable of pumping Ca2+ ions, under physiological conditions, with variable stoichiometry that is dependent upon its thermodynamic loading.     

Ca2+-ATPases (SERCA): energy transduction and heat production in transport ATPases

The sarcoplasmic reticulum Ca2+-ATPase is able to cleave ATP through two different catalytic routes. In one of them, a part of the chemical energy derived from ATP hydrolysis is used to transport Ca2+ across the membrane and part is dissipated as heat. In the second route, the hydrolysis of ATP is completed before Ca2+ transport and all the energy derived from ATP hydrolysis is converted into heat. The second route is activated by the rise of the Ca2+ concentration in the vesicle lumen. In vesicles derived from white skeletal muscle the rate of the uncoupled ATPase is several-fold faster than the rate of the ATPase coupled to Ca2+ transport, and this accounts for both the low Ca2+/ATP ratio usually measured during transport and for the difference of heat produced during the hydrolysis of ATP by intact and leaky vesicles. Different drugs were found to selectively inhibit the uncoupled ATPase activity without modifying the activity coupled to Ca2+ transport. When the vesicles are actively loaded, part of the Ca2+ accumulated leaks to the medium through the ATPase. Heat is either produced or released during the leakage, depending on whether or not the Ca2+ efflux is coupled to the synthesis of ATP from ADP and Pi.     

Voltage-dependence of Ca2+ uptake and ATP hydrolysis of reconstituted Ca2+-ATPase vesicles

Ca2+-ATPase from sarcoplasmic reticulum was reconstituted into phospholipid/cholesterol (9:1) vesicles (RO). Sucrose density gradient centrifugation of the RO vesicles separated a light layer (RL) with a high lipid/protein ratio and a heavy layer (RH). RH vesicles exhibited a high rate of Ca2+-dependent ATP hydrolysis but did not accumulate Ca2+. RL vesicles, on the other hand, showed an initial molar ratio of Ca2+ uptake to ATP hydrolysis of approximately 1.0. Internal trapping of transported Ca2+ facilitated studies over periods of several minutes. Ca2+ transport and ATP hydrolysis declined concomitantly, reaching levels near 0 with external Ca2+ concentrations less than or equal to 2 microM. Ca2+ uptake was inhibited by the Ca2+ ionophore A23187, the detergent Triton X-100, and the metabolic inhibitor quercetin. Ca2+ transport generated a transient electrical potential difference, inside positive. This finding is consistent with the hypothesis that the Ca2+ pump is electrogenic. Steady state electrical potentials across the membrane were clamped by using potassium gradients and valinomycin, and monitored with voltage-sensitive dyes. Over a range of +50 to -100 mV, there was an inverse relationship between the initial rate of Ca2+ uptake and voltage, but the rate of ATP hydrolysis was nearly constant. In contrast, lowering the external Ca2+ concentration depressed both transport and ATP hydrolysis. These findings suggest that the membrane voltage influences the coupling between Ca2+ transport and ATP hydrolysis.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37 degrees C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 microM CaCl2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca2+ uptake, a second phase of Ca2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca2+ influx of sarcoplasmic reticulum near steady state of Ca2+ uptake was measured by pulse labeling with 45Ca2+. The Ca2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca2+ uptake in normal medium, Ca2+ influx was balanced by Ca2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca2+ exchange rate at the first plateau of Ca2+ uptake was about half of that in normal medium. When the second phase of Ca2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 micrograms/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca2+ uptake was also observed. These data suggest that the Ca2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca2+ efflux, which subsequently stimulates Ca2+ exchange.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

ATP reversible Pi exchange and membrane phosphorylation in sarcoplasmic reticulum vesicles: activation by silver in the absence of a Ca2+ concentration gradient.

The activation of ATP reversible Pi exchange, normally associated with a Ca2+ concentration gradient in sarcoplasmic reticulum vesicles, can be obtained in "leaky" vesicles in 4-10 mM CaCl2. In the micromolar range, Ag+ activates the ATP reversible Pi exchange two- to fourfold. Similar concentrations of Ag+ promote a parallel inhibition of Ca2+- activated ATP hydrolysis and Ca2+ uptake in intact vesicles. Maximal inhibition of these activities by Ag+ leaves the Mg2+-dependent ATPase unaffected. No net synthesis of ATP was demonstrated in leaky vesicles. The effects of Ag+ depends on the protein concentration and persist after removal of Ag+ from the medium. Membrane phosphorylation from Pi or from ATP is respectively activated or inhibited by Ag+ in reciprocal fashion.     

Reaction mechanism of (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles. II. (ATP, ADP)-dependent Ca2+-Ca2+ exchange across the membranes

Sarcoplasmic reticulum vesicles were preloaded with unlabeled CaCl2, and 45Ca2+ incorporation into the vesicles was determined by adding 45CaCl2 to the external medium in the presence of ATP and ADP. In the absence of added MgCl2, the steady state rate of the (ATP, ADP)-dependent 45Ca2+ incorporation was extremely low, being in good agreement with that of the Ca2+-dependent ATP hydrolysis which was catalyzed by the membrane-bound (Ca2+, Mg2+)-ATPase. In contrast, it was greatly increased by addition of MgCl2 and became much higher than the steady state rate of the Ca2+-dependent ATP hydrolysis. The kinetic analysis of the results gave support to the probability that the MgCl2 addition markedly shifted the equilibrium of the reaction of Caout . EP and Cain . EP represent phosphoenzymes with bound Ca2+ which is exposed to the external medium and to the internal medium, respectively).     

Effects of nonsolubilizing and solubilizing concentrations of Triton X-100 on Ca2+ binding and Ca2+-ATPase activity of sarcoplasmic reticulum

The effect of low concentrations of Triton X-100, below that required for solubilization, on the properties of the Ca2+-ATPase of sarcoplasmic reticulum has been investigated. The changes observed have been compared with the changes produced on solubilization of the vesicles at higher concentrations of detergent. In the range 0.02-0.05% (w/v) Triton X-100, concentrations which did not solubilize the vesicles but completely inhibit ATP-mediated Ca2+ accumulation, 8-16 mol of detergent/mol of ATPase was associated with the vesicles. This amount of Triton X-100 altered equilibrium Ca2+ binding and Ca2+ activation of p-nitrophenyl phosphate and of ATP hydrolysis in a manner which lowered the apparent Ca2+ cooperatively (nH = 1 or less), and which increased the K0.5(Ca) value 20-fold. These changes in Ca2+ binding and activation parameters were associated with a 90% lower Ca2+-induced change in fluorescence of fluorescein isothiocyanate modified enzyme. The rates of p-nitrophenyl phosphate and of ATP hydrolysis, at saturating Ca2+ concentrations, were about half that of detergent-free vesicles. The rate constant for phosphoenzyme hydrolysis in the absence of Ca2+, calculated from medium Pi in equilibrium HOH exchange and phosphoenzyme measurements, was lowered from 38 to 11 s-1. The steady-state level of phosphoenzyme formed from Pi in the absence of Ca2+ was slightly increased up to 0.02% Triton X-100 and then decreased about half at 0.05%. The synthesis of ATP in single turnover type experiments was not affected by detergent binding. Pi in equilibrium ATP exchange was inhibited 65%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coincidence of H+ binding and Ca2+ dissociation in the sarcoplasmic reticulum Ca-ATPase during ATP hydrolysis

H+ and Ca2+ concentration changes in the reaction medium following MgATP addition at pH 6.0 were determined with the partially purified Ca-ATPase from sarcoplasmic reticulum vesicles in the presence of 25-50 microM CaCl2 and 5 mM MgCl2 at 4 degrees C. Previously, we showed a sequential occurrence of H+ binding and H+ dissociation in the Ca-ATPase during ATP hydrolysis and further suggested that the H+ binding takes place inside the vesicles (Yamaguchi, M., and Kanazawa, T. (1984) J. Biol. Chem. 259, 9526-9531). The present results demonstrate that the H+ binding occurred coincidently with Ca2+ dissociation from the enzyme upon conversion of the phosphoenzyme (EP) intermediate from the ADP-sensitive form to the ADP-insensitive form in the catalytic cycle of ATP hydrolysis. As KCl decreased in the medium, the extent of the H+ binding increased almost proportionately with the extent of either the Ca2+ dissociation or the accumulation of ADP-insensitive EP. Both the H+ binding and the Ca2+ dissociation were prevented by a modification of the specific SH group of the enzyme essential for the conversion of ADP-sensitive EP to ADP-insensitive EP. In the late stage of the reaction, H+ dissociation from the enzyme occurred coincidently with Ca2+ binding to the dephosphoenzyme which was formed by EP decomposition. These results are consistent with the possibility that the H+ ejection during the Ca2+ uptake with the intact vesicles previously shown by several investigators takes place through a Ca2+/H+ exchange directly mediated by the membrane-bound Ca-ATPase.     

Ca2+ translocation and catalytic activity of the sarcoplasmic reticulum ATPase. Modulation by ATP, Ca2+, and Pi

The ratio between Ca2+ uptake and Ca(2+)-dependent ATP hydrolysis measured in sarcoplasmic reticulum vesicles of rabbit skeletal muscle was found to vary greatly depending on the concentrations of oxalate or Pi used. In the presence of 5 mM oxalate, 20 mM Pi, and 1 mM Pi, the ratios found were in the range of 1.4-2.3, 0.6-0.8, and 0.01-0.10, respectively. The rates of Ca2+ exchange and ATP synthesis were measured at the steady state by adding trace amounts of 45Ca and 32Pi, after the vesicles had been loaded with Ca2+. In the presence of 1 mM Pi, 10 mM MgCl2, and 0.2 mM CaCl2, the ratio between Ca2+ exchange and ATP synthesis varied from 9 to 14. This ratio approached two when Ca2+ in the medium was reduced to a very low level, or when in the presence of Ca2+, dimethyl sulfoxide was added to the assay medium, or when the Pi concentration was raised from 1 to 20 mM. A ratio of two was also measured when the steady state was attained using ITP instead of ATP. In all the conditions that led to a ratio close to two, there was an increase in the fraction of enzyme phosphorylated by Pi. It is proposed that the coupling between Ca2+ translocation and ATP hydrolysis or synthesis is modulated by the phosphorylation of the ATPase by Pi.     

ATP regulation of calcium transport in back-inhibited sarcoplasmic reticulum vesicles

At high concentrations of ATP, ATP hydrolysis and Ca2+ transport by the (Ca2+ + MG2+)-ATPase of intact sarcoplasmic reticulum vesicles exhibit a secondary activation that varies with the extent of back-inhibition by Ca2+ accumulated within the vesicles. When the internal ionized Ca2+ is clamped at low and intermediate levels by the use of Ca-precipitating anions, the apparent Km values for activation by ATP are lower than in fully back-inhibited vesicles (high internal Ca2+). In leaky vesicles unable to accumulate Ca2+, raising Ca2+ in the assay medium from 20-30 microM to 5 mM abolishes the activation of hydrolysis by high concentrations of ATP. The level of [32P]phosphoenzyme formed during ATP hydrolysis from [32P]phosphate added to the medium also varies with the extent of back-inhibition; it is highest when Ca2+ is raised to a level that saturates the internal, low-affinity Ca2+ binding sites. In intact vesicles, increasing the ATP concentration from 10 to 400 microM competitively inhibits the reaction of inorganic phosphate with the enzyme but does not change the rate of hydrolysis. In a previous report (De Meis, L., Gomez-Puyou, M.T. and Gomez-Puyou, A. (1988) Eur. J. Biochem. 171, 343-349), it has been shown that the hydrophobic molecules trifluoperazine and iron bathophenanthroline compete for the catalytic site of the Pi-reactive form of the enzyme. Here it is shown that inhibition of ATP hydrolysis by these compounds is reduced or abolished when Ca2+ binds to the low-affinity Ca2+ binding sites of the enzyme. Since inhibition by these agents is indifferent to activation of hydrolysis by high concentrations of ATP, it is suggested that the second Km for ATP and the inhibition by hydrophobic molecules involve two different Ca-free forms of the enzyme.     

ATP-dependent calcium transport in plasma membrane vesicles from neutrophil leukocytes

Plasma membrane vesicles were prepared from guinea pig peritoneal exudate neutrophils, using nitrogen cavitation to rupture the plasma membrane and differential centrifugation to separate the vesicles. The vesicles were enriched 13.2-fold in (Na+, K+)-ATPase activity and had a cholesterol:protein ratio of 0.15, characteristic of plasma membranes. Contamination of the vesicle preparation with DNA or marker enzyme activities for intracellular organelles was very low. Studies designed to determine vesicle sidedness and integrity indicated that 33% were sealed, inside-out; 41% were sealed, right side-out, and 26% were leaky. The vesicles accumulated 45Ca2+ in a linear fashion for 45 min. The uptake was dependent on the presence of oxalate and MgATP in the incubating medium. Uptake showed a Ka for free Ca2+ of 164 nM and a Vmax of 17.2 nmol/mg . min (based on total protein). GTP, ITP, CTP, UTP, ADP, or AMP supported uptake at rates less than or equal to 11% of ATP. Ca2+ uptake was maximal at pH 7-7.5. Calcium stimulated the hydrolysis of ATP by the vesicles with a Ka for free Ca2+ of 440 nM and Vmax of 17.5 nmol/mg . min (based on total protein). When the Ca2+ uptake rate was based upon those vesicles expected to transport Ca2+ (33% sealed, inside-out vesicles) and Ca2+-stimulated ATPase activity was based upon those vesicles expected to express that activity (26% leaky + 33% sealed, inside-out vesicles), the molar stoichiometry of Ca2+ transported:ATP hydrolyzed was 2.12 +/- 0.12. Calmodulin did not increase either Vmax or Ka for free Ca2+ of the uptake system in the vesicles, even when they were treated previously with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The high affinity of this system for Ca2+, specificity for ATP, physiological pH optimum, and stoichiometry of Ca2+ transported:ATP hydrolyzed suggest that it represents an important mechanism by which neutrophils maintain low levels of cytoplasmic free Ca2+.     

Magnesium permeability of sarcoplasmic reticulum. Mg2+ is not countertransported during ATP-dependent Ca2+ uptake by sarcoplasmic reticulum

Magnesium transport across sarcoplasmic reticulum (SR) vesicles was investigated in reaction mixtures of various composition using antipyrylazo III or arsenazo I to monitor extravesicular free Mg2+. The half-time of passive Mg2+ efflux from Mg2+-loaded SR was 100 s in 100 mM KCl, 150 S in 100 mM K gluconate, and 370 S in either 100 mM Tris methanesulfonate or 200 mM sucrose solutions. The concentration and time course of Mg2+ released into the medium was also measured during ATP-dependent Ca2+ uptake by SR. In reaction mixtures containing up to 3 mM Mg2+, small changes in free magnesium of 10 microM or less were accurately detected without interference from changes in free Ca2+ of up to 100 microM. Three experimental protocols were used to determine whether the increase of free [Mg2+] in the medium after an addition of ATP was due to Mg2+ dissociated from ATP following ATP hydrolysis or to Mg2+ translocation from inside to outside of the vesicles. 1) In the presence of ATP-regenerating systems which maintained constant ATP to ADP ratios and normal rates of active Ca2+ uptake, the increase of Mg2+ in the medium was negligible. 2) Mg2+ released during ATP-dependent Ca2+ uptake by SR was similar to that observed during ATP hydrolysis catalyzed by apyrase, in the absence of SR. 3) In SR lysed with Triton X-100 such that Ca2+ transport was uncoupled from ATPase activity, the rate and amount of Mg2+ release was greater than that observed during ATP-dependent Ca2+ uptake by intact vesicles. Taken together, the results indicate that passive fluxes of Mg2+ across SR membranes are 10 times faster than those of Ca2+ and that Mg2+ is not counter-transported during active Ca2+ accumulation by SR even in reaction mixtures containing minimal concentrations of membrane permeable ions that could be rapidly exchanged or cotransported with Ca2+ (e.g. K+ or Cl-).     

Calcium and magnesium regulation of phosphorylation by ATP and ITP in sarcoplasmic reticulum vesicles.

Membrane phosphorylation and nucleoside triphosphatase activity of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were studied using ATP and ITP as substrates. The Ca2+ concentration was varied over a range large enough to saturate either the high affinity Ca2+-binding site or both high and low affinity binding sites. In intact vesicles, which are able to accumulate Ca2+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca2+ concentration is increased to 10 mM. Essentially the same pattern of membrane phosphorylation by ATP is observed when leaky vesicles, which are unable to accumulate Ca2+, are used. However, for leaky vesicles, when ITP is used as substrate, the phosphoenzyme level increases 3- to 4-fold when the Ca2+ concentration is raised from 0.02 to 20 mM. When Mg2+ is omitted from the assay medum, the degree of membrane phosphorylation by ATP varies with Ca2+ in the same way as when ITP is used in the presence of Mg2+. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added Mg2+. When these vesicles are incubated in media containing ITP and 0.1 mM Ca2+, addition of Mg2+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When ATP is used, the addition of 10 mM Mg2+ increases both the steady state level of phosphoenzyme and the rate of ATP hydrolysis. When the Ca2+ concentration is raised to 10 or 20 mM, the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg2+. In these conditions the ATPase and ITPase activities are activated by Mg2+, although not to the level observed in 0.1 mM Ca2+. An excess of Mg2+ inhibits both the rate of hydrolysis and membrane phosphorylation by either ATP or ITP.     

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.     

Coupling of Ca2+ transport to ATP hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum: potential role of the 53-kilodalton glycoprotein

An essential feature of the function of the Ca2+-ATPase of sarcoplasmic reticulum (SR) is the close coupling between the hydrolysis of ATP and the active transport of Ca2+. The purpose of this study is to investigate the role of other components of the SR membrane in regulating the coupling of Ca2+-ATPase in SR isolated from rabbit skeletal muscle, reconstituted SR, and purified Ca2+-ATPase/phospholipid complexes. Our results suggest that (1) it is possible to systematically alter the degree of coupling obtained in reconstituted SR preparations by varying the [KC1] present during cholate solubilization, (2) the variation in coupling is not due to differences in the permeability of the reconstituted SR vesicles to Ca2+, and (3) vesicles reconstituted with purified Ca2+-ATPase are extensively uncoupled under our experimental conditions regardless of the lipid/protein ratio or phospholipid composition. In reconstituted SR preparations prepared by varying the [KC1] present during cholate treatment, we find a direct correlation between the relative degree of coupling between ATP hydrolysis and Ca2+ transport and the level of the 53-kilodalton (53-kDa) glycoprotein of the SR membrane. These results suggest that the 53-kDa glycoprotein may be involved in regulating the coupling between ATP hydrolysis and Ca2+ transport in the SR.     

Role of Mg2+ in the Ca2+-Ca2+ exchange mediated by the membrane-bound (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum vesicles were preloaded with either 45Ca2+ or unlabeled Ca2+. The unidirectional Ca2+ efflux and influx, together with Ca2+-dependent ATP hydrolysis and phosphorylation of the membrane-bound (Ca2+, Mg2+)-ATPase, were determined in the presence of ATP and ADP. The Ca2+ efflux depended on ATP (or ADP or both). It also required the external Ca2+. The Ca2+ concentration dependence of the efflux was similar to the Ca2+ concentration dependences of Ca2+ influx, Ca2+-dependent ATP hydrolysis, and phosphoenzyme formation. The rate of the efflux was approximately in proportion to the concentration of the phosphoenzyme up to 10 microM Ca2+. These results and other findings indicate that the Ca2+ efflux represents the Ca2+-Ca2+ exchange (between the external medium and the internal medium) mediated by the phosphoenzyme. In the range of 0.6-5.2 microM Mg2+, no appreciable Ca2+-Ca2+ exchange was detected although phosphoenzyme formation occurred to a large extent. Elevation of Mg2+ in the range 5.2 microM-4.8 mM caused a remarkable activation of the exchange, whereas the amount of the phosphoenzyme only approximately doubled. The kinetic analysis shows that this activation results largely from the Mg2+-induced acceleration of an exchange between the bound Ca2+ of the phosphoenzyme and the free Ca2+ in the internal medium. It is concluded that Mg2+ is essential for the exposure of the bound Ca2+ of the phosphoenzyme to the internal medium.     

Calcium dependence during single-cycle catalysis of the sarcoplasmic reticulum ATPase

We have investigated the kinetic and thermodynamic properties of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum under conditions that result in a single transport cycle. Simultaneous addition of ATP and EGTA to sarcoplasmic reticulum vesicles, preincubated with calcium, resulted in a transient of intermediate species. In the presence of saturating Ca2+ levels, total E-P species reached a maximum of 2.3 nmol/mg at 100 ms, followed by a monoexponential decay with kobs = 3.6 s-1. The data are interpreted in terms of Ca2+ sequestration, either by occlusion as Ca2+ in the phosphorylated enzyme or chelation by EGTA. Maximum Ca2+ uptake was 8.3 nmol/mg with the release of 4.4 nmol/mg Pi. The ratio of Ca2+ uptake to Pi release approached 1.9 over a wide [Ca2+] range. Equilibrium Ca2+ binding, in the absence of ATP, showed a K0.5 of 0.88 microM with a Hill coefficient of 1.9. The Ca2+ concentration dependence of Ca2+ uptake during single-cycle catalysis showed a 10-fold enhanced affinity (K0.5 = 0.06 microM) and was noncooperative (nH = 0.9). Quench with excess EGTA (greater than 2 mM) decreased Ca2+ uptake to 1 nmol/mg, indicating an "off" rate of Ca2+ from high affinity sites that exceeds 100 s-1. The ATP concentration dependence for a single-cycle catalysis showed an apparent K0.5 of 1.1 microM, similar to that for ATP equilibrium binding. It is proposed that enzyme phosphorylation proceeds only following binding of a second calcium ion to externally oriented sites whose intrinsic affinity is in the same range as the calcium dependence of a single-cycle turnover.     

Interaction of asparagine-linked oligosaccharides with an immobilized rice (Oryza sativa) lectin column.

Inside-out plasma-membrane vesicles isolated from rat liver [Prpic, Green, Blackmore & Exton (1984) J. Biol. Chem. 259, 1382-1385] accumulated a substantial amount of 45Ca2+ when they were incubated in a medium whose ionic composition and pH mimicked those of cytosol and which contained MgATP. The Vmax of the initial 45Ca2+ uptake rate was 2.9 +/- 0.6 nmol/min per mg and the Km for Ca2+ was 0.50 +/- 0.08 microM. The ATP-dependent 45Ca2+ uptake by inside-out plasma-membrane vesicles was about 20 times more sensitive to saponin than was the ATP-dependent uptake by a microsomal preparation. The 45Ca2+ efflux from the inside-out vesicles, which is equivalent to the Ca2+ influx in intact cells, was increased when the free Ca2+ concentration in the medium was decreased. The Ca2+ antagonists La3+ and Co2+ inhibited the 45Ca2+ efflux from the vesicles. Neomycin stimulated the Ca2+ efflux in the presence of either a high or a low free Ca2+ concentration. These results confirm that polyvalent cations regulate Ca2+ fluxes through the plasma membrane.