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• W2000894098 abstract "Once two radioactive Ca2+ coming from the cytoplasm are bound to the transport sites of the nonphosphorylated ATPase, excess EGTA induces rapid dissociation of both ions, whereas excess nonradioactive Ca2+ only reaches one of the two bound Ca2+. This difference has been explained assuming that the two Ca2+ sites are in a single file channel in which the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is exchangeable only when the superficial site is vacant. The same experiment was done using phosphorylated ATPase to determine whether Ca2+ dissociation toward the lumen is sequential as well. Under conditions that allow ADP-sensitive phosphoenzyme to accumulate (leaky vesicles, 5°C, pH 8, 300 mM KCl), we found the same two pools of Ca2+. Excess EGTA induced dissociation of both ions together with dephosphorylation. Excess nonradioactive Ca2+ induced the exchange of half the radioactive Ca2+ without any effect on the phosphoenzyme level. Our results show a close similarity between the transport sites of the nonphosphorylated and the phosphorylated enzymes, although the orientation, affinities, and dissociation rate constants are different. Once two radioactive Ca2+ coming from the cytoplasm are bound to the transport sites of the nonphosphorylated ATPase, excess EGTA induces rapid dissociation of both ions, whereas excess nonradioactive Ca2+ only reaches one of the two bound Ca2+. This difference has been explained assuming that the two Ca2+ sites are in a single file channel in which the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is exchangeable only when the superficial site is vacant. The same experiment was done using phosphorylated ATPase to determine whether Ca2+ dissociation toward the lumen is sequential as well. Under conditions that allow ADP-sensitive phosphoenzyme to accumulate (leaky vesicles, 5°C, pH 8, 300 mM KCl), we found the same two pools of Ca2+. Excess EGTA induced dissociation of both ions together with dephosphorylation. Excess nonradioactive Ca2+ induced the exchange of half the radioactive Ca2+ without any effect on the phosphoenzyme level. Our results show a close similarity between the transport sites of the nonphosphorylated and the phosphorylated enzymes, although the orientation, affinities, and dissociation rate constants are different. Sarcoplasmic reticulum Ca2+-ATPase is a membranous enzyme that pumps Ca2+ from the cytoplasm of muscle cells into the reticulum lumen, requiring ATP hydrolysis. The ATPase cycle transports 2Ca2+ per molecule of ATP and per monomer of ATPase, as described in Fig. S1. During the cycle, the transport sites change their orientation and affinity, depending on whether the ATPase is phosphorylated. The high-affinity transport sites of the nonphosphorylated ATPase are accessible from the cytoplasm, whereas once the ATPase has been phosphorylated the transport sites have lower affinity and are accessible from the lumen. This allows Ca2+ release into the SR 1The abbreviations used are: SRsarcoplasmic reticulumTesN-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acidA23187calcimycin. lumen and is followed by dephosphorylation of the phosphoenzyme. sarcoplasmic reticulum N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid calcimycin. Ca2+ binding to E, the Ca2+-deprived nonphosphorylated ATPase, has been well characterized. Two ions bind sequentially with a high affinity and a positive cooperativity which both depend on the experimental conditions, Ca = 0.1-10 μM and nH = 1.3-2, for pH 8-6, and 0-3 mM Mg2+ at 20°C (1Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10953-10960Abstract Full Text PDF PubMed Google Scholar). The two Ca2+ ions are known to be kinetically distinguishable, since Dupont (2Dupont Y. Biochim. Biophys. Acta. 1982; 688: 75-87Crossref PubMed Scopus (88) Google Scholar) showed that the dissociation of half the 45Ca2+ bound to E was impaired by the presence of excess 45Ca2+ in the medium. This has been confirmed by several authors under various experimental conditions, different temperatures, pH, Mg2+ concentrations …(3Dupont Y. Anal. Biochem. 1984; 142: 504-510Crossref PubMed Scopus (59) Google Scholar, 4Nakamura J. Biochim. Biophys. Acta. 1986; 870: 495-501Crossref PubMed Scopus (14) Google Scholar, 5Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar, 6Petithory J.R. Jencks W.P. Biochemistry. 1988; 27: 5553-5564Crossref PubMed Scopus (63) Google Scholar, 7Orlowski S. Champeil P. Biochemistry. 1991; 30: 352-361Crossref PubMed Scopus (77) Google Scholar, 8Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10961-10968Abstract Full Text PDF PubMed Google Scholar). A simple example of two sites being sequentially accessible from the cytoplasm by the two Ca2+ ions is a channel with a deep site and a superficial site (see the sketch in Fig. 2). The first ion must reach the deep site to leave the superficial site vacant for the second ion. The Ca2+ bound to the superficial site is freely exchangeable with the outer medium, whereas the Ca2+ bound to the deep site is not. Such a description of a channel-like structure for the transport sites was first proposed by Inesi(5Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar). Inesi (5Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar) took advantage of the possibility to selectively place one 45Ca2+ on top of one 45Ca2+, to determine whether their dissociation toward the lumen is sequential. By monitoring the internalization of the Ca2+ ions after phosphorylation, Inesi (5Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar) concluded that their dissociation toward the lumen is sequential and that the first Ca2+ bound to E is the first to be internalized. The question whether the dissociation of the Ca2+ ions toward the lumen is sequential or not has been reinvestigated more recently by Hanel and Jencks (9Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar) and Orlowski and Champeil(10Orlowski S. Champeil P. Biochemistry. 1991; 30: 11331-11342Crossref PubMed Scopus (37) Google Scholar). In observing the dissociation of Ca2+ from the phosphorylated ATPase, Orlowski and Champeil (10Orlowski S. Champeil P. Biochemistry. 1991; 30: 11331-11342Crossref PubMed Scopus (37) Google Scholar) found no difference between the dissociation kinetics induced by EGTA or cold Ca2+ and no difference in the dissociation rates of each individual ion. Hanel and Jencks (9Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar) found no difference between the Ca2+ internalization rates observed using empty vesicles, or vesicles loaded with 20 mM Ca2+, and also no difference between the internalization rates of each individual ion. In both papers, the authors concluded that the two ions cannot be kinetically distinguished. The explanation given by Hanel and Jencks (9Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar) is that a slow conformational change corresponding to deocclusion of Ca2+ precedes fast dissociation of Ca2+ from the phosphoenzyme, therefore making the measurement of the individual Ca2+ dissociation steps impossible. The discrepancy between the results of Inesi (5Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar) on the one hand and those of Hanel and Jencks (9Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar) and Orlowski and Champeil (10Orlowski S. Champeil P. Biochemistry. 1991; 30: 11331-11342Crossref PubMed Scopus (37) Google Scholar) on the other hand is difficult to understand, because they all used similar experimental conditions, typically pH 6.8-7.5, room temperature, 80-100 mM KCl. Here we show that with 300 mM KCl, at pH 8 and 5°C, the lumenal dissociation of the two Ca2+ ions from the phosphorylated ATPase is sequential, as is the cytoplasmic dissociation of the two Ca2+ ions from the nonphosphorylated ATPase. SR vesicles were prepared and tested as described in (1Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10953-10960Abstract Full Text PDF PubMed Google Scholar) from rabbits subjected to a 48-h starvation diet to lower the contamination by phosphorylase(11Cuenda A. Centeno F. Gutierrez-Merino C. FEBS Lett. 1991; 283: 273-276Crossref PubMed Scopus (22) Google Scholar). All experiments were carried out at 5°C in a cold room, and the buffer was always 100 mM Tes-Tris, pH 8, 300 mM KCl. It was prepared with water filtered through a Milli-Q Water Purification System (Millipore). All salts were added as chlorides. Vesicles were made leaky by an incubation of at least 1 h at 2 mg/ml in 50 mM Tris, 10 mM KCl, 2 mM EDTA, at room temperature. Kinetic measurements involving [45Ca]Ca2+ or [γ-32P]ATP all started with the same incubation and rinsing steps. Vesicles (0.2 mg/ml) were first incubated in the pH 8 buffer, plus Mg2+, as specified. 1 ml of this suspension was deposited on a filter (Millipore HA 0.45), and the adsorbed vesicles were rinsed with 1 ml of 100 μM EGTA to deprive the enzyme of contaminating Ca2+. For cytoplasmic Ca2+ dissociation experiments, ATPase was converted to the Ca2E state by manually perfusing the filters for 5 s with 1 ml of 10 μM [45Ca]Ca2+. For lumenal Ca2+ dissociation experiments, ATPase was converted to the Ca2E-P state by manually perfusing the filters for 5 s with 2 ml of 100 μM [45Ca]Ca2+ or 45Ca2+, 100 μM [γ-32P]ATP or ATP, and Mg2+ as specified. The kinetic measurements were started immediately after this step using a rapid filtration apparatus (Biologic, Claix, France). They were done by perfusing 1 mM EGTA, or 1 mM45Ca2+, plus Mg2+ as specified, for various times. All solutions containing [45Ca]Ca2+ or [γ-32P]ATP also contained 1 mM [3H]glucose, which allows evaluation of the filter wet volume, usually about 30 μl. 3H and 45Ca or 32P retained on the filter were simultaneously measured by scintillation. [γ-32P]ATP or [45Ca]Ca2+ contained in the wet volume was subtracted from the total 32P or 45Ca counts to evaluate the phosphoenzyme and the Ca2+ bound to the ATPase. [45Ca]Ca2+ accumulation in the vesicles was measured in the presence of [45Ca]Ca2+, [3H]glucose, ATP, and Mg2+ as specified. The reaction was started by addition of vesicles at 0.2 mg/ml. After various periods of time, 1 ml of the reaction mixture was deposited on a filter (Millipore HA 0.45), and the radioactivity retained on the filter was counted. Steady-state ATPase activity was measured spectrophotometrically by coupling ATP hydrolysis to NADH oxidation in the presence of 0.4 mg/ml pyruvate kinase, 0.2 mg/ml lactate dehydrogenase, 1 mM phosphoenolpyruvate, 0.45 mM NADH, 1-3 mM Mg2+, 0.1-1 mM ATP, 300 mM KCl, at pH 8 and 6-10°C. NADH absorbance variations were followed at 350 nm by an HP 8452A diode array spectrophotometer. If it is assumed that the dissociation of the two Ca2+ ions from the phosphorylated ATPase is intrinsically sequential, the fact that Inesi (5Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar) found that the two ions dissociate sequentially, whereas Hanel and Jencks (9Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar) and Orlowski and Champeil (10Orlowski S. Champeil P. Biochemistry. 1991; 30: 11331-11342Crossref PubMed Scopus (37) Google Scholar) found that the two ions could not be distinguished can simply indicate that this assumption was difficult to prove under their approximately similar conditions. Keeping this in mind, we looked for experimental conditions that would allow measuring an effect of cold Ca2+ on the lumenal dissociation of radioactive Ca2+ bound to the phosphorylated ATPase. Alkaline pH has been shown to increase the affinity of the phosphorylated ATPase for Ca2+(12Verjovski-Almeida S. de Meis L. Biochemistry. 1977; 16: 329-334Crossref PubMed Scopus (31) Google Scholar) and to be even more effective at low temperatures(13de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar). Also high KCl concentrations are known to favor Ca2E-P, the ADP-sensitive phosphoenzyme(14Shigekawa M. Akowitz A.A. J. Biol. Chem. 1979; 254: 4726-4730Abstract Full Text PDF PubMed Google Scholar). Thus, 300 mM KCl, pH 8 and 5°C, were the conditions chosen for these experiments, as we expected to have all the ATPase in its Ca2E-P form, with a low ATPase activity and a high enough affinity for lumenal Ca2+ to see an effect of cold Ca2+ on the dissociation of radioactive Ca2+. Ca2+ accumulation in the vesicles and steady-state ATPase activity were measured under the conditions of the Ca2+ dissociation experiments, i.e. 300 mM KCl, pH 8 at 5°C, to check the activity of the enzyme under such conditions. Fig. 1 shows Ca2+ accumulation into the vesicles during 30-min incubation in the presence of 100 μM ATP. With 3 mM Mg2+, tight vesicles accumulated 70 nmol/mg, a value that is comparable with the maximum amount of Ca2+ accumulated into the vesicles under standard conditions (i.e. 80 nmol/mg at pH 6 and 20°C), and the so-called leaky vesicles did not accumulate Ca2+, as was intended. In the absence of Mg2+, accumulation was lower, as expected from slower turnover in the presence of CaATP(15Yamada S. Ikemoto N. J. Biol. Chem. 1980; 255: 3108-3119Abstract Full Text PDF PubMed Google Scholar). Steady-state ATPase activity was measured as described above, using tight vesicles permeabilized by 4% (w/w) A23187 or leaky vesicles. The steady-state activity measured in the presence of 3 mM Mg2+ and 100 μM ATP was 240 nmol/mg/min at 8°C. Once two radioactive Ca2+ coming from the cytoplasm have been bound to the transport sites of the nonphosphorylated ATPase, excess EGTA induces rapid dissociation of both ions, whereas excess nonradioactive Ca2+ induces rapid dissociation of only one of the two bound Ca2+(2Dupont Y. Biochim. Biophys. Acta. 1982; 688: 75-87Crossref PubMed Scopus (88) Google Scholar). This rapidly exchangeable Ca2+ has been identified as the last Ca2+ bound to ATPase(5Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar, 6Petithory J.R. Jencks W.P. Biochemistry. 1988; 27: 5553-5564Crossref PubMed Scopus (63) Google Scholar). As the dissociation of the other Ca2+ is impaired by the binding of cold Ca2+ at the exchangeable site, it has been identified as the first Ca2+ bound to ATPase(6Petithory J.R. Jencks W.P. Biochemistry. 1988; 27: 5553-5564Crossref PubMed Scopus (63) Google Scholar). The two sites are sketched in Fig. 2, which also reports the results of cytoplasmic Ca2+ dissociation experiments under the present conditions, namely using leaky vesicles at pH 8 and 5°C, in the presence of 300 mM KCl. Ca2E was formed by perfusing the vesicles with 10 μM [45Ca]Ca2+ plus 3 mM Mg2+, and dissociation was initiated by perfusing 1 mM EGTA or 1 mM45Ca2+, plus 3 mM Mg2+. EGTA induced biphasic dissociation of Ca2+ ions with rates of 20 and 1 s-1, whereas 45Ca2+ induced dissociation of only half the radioactive Ca2+ with the rate of 9 s-1. This experiment was repeated using tight vesicles that yielded similar rates for cytoplasmic Ca2+ dissociation (data not shown), indicating that the Ca2+ transport sites are not modified by the incubation in EDTA and Tris. Phosphorylated ATPase was formed by perfusing tight vesicles with 100 μM [45Ca]Ca2+, 100 μM [γ-32P]ATP, 3 mM Mg2+, 300 mM KCl for 5 s. It was then perfused with 1 mM EGTA plus 3 mM Mg2+ (Fig. 3, open symbols). The amounts of bound [45Ca]Ca2+ and phosphoenzyme were unchanged even after 10 s, indicating that Ca2+ was no longer accessible from the cytoplasmic side. This so-called occluded Ca2+ can be either occluded in the membrane in the Ca2E-P form of ATPase as the Ca2+ bound to this form is not accessible from the cytoplasmic side or it can be occluded inside the vesicles, if ATPase turnover is such that some Ca2+ has been transported during the phosphorylation step(16Dupont Y. Eur. J. Biochem. 1980; 109: 231-238Crossref PubMed Scopus (123) Google Scholar, 17Takisawa H. Makinose M. Nature. 1981; 290: 271-273Crossref PubMed Scopus (37) Google Scholar). The existence of these two types of occluded Ca2+ is demonstrated by the effect of ADP, as shown in Fig. 3(closed symbols). Almost all the occluded Ca2+ was trapped in the membrane in the Ca2E-P form of ATPase. Phosphorylated ATPase, prepared as described above, was perfused with a mixture of 300 μM ADP and 1 mM EGTA. At variance with the perfusion with 1 mM EGTA alone, with ADP present there was fast dephosphorylation together with Ca2+ dissociation. This experiment showed that all the phosphoenzyme was ADP sensitive, as expected from the use of 300 mM KCl at low temperature, and that there was 8 nmol/mg calcium accumulated in the vesicle lumen during the phosphorylation step. Phosphorylated ATPase was formed as above, but using leaky vesicles. It was then perfused with 1 mM EGTA plus 3 mM Mg2+ (Fig. 4) or 1 mM45Ca2+ plus 3 mM Mg2+ (Fig. 5). The perfusion with EGTA induced biphasic dissociation of all the bound [45Ca]Ca2+, together with dephosphorylation of ATPase at rates of 0.6 and 0.06 s-1, whereas the perfusion with 45Ca2+ induced biphasic dissociation of [45Ca]Ca2+ at 0.4 and 0.03 s-1 and slow dephosphorylation at 0.03 s-1. To determine whether the phosphoenzyme measured during these perfusions was still the Ca2E-P form of ATPase, its ADP sensitivity was tested at time 0 and after 19 s of perfusion. That is the perfusions with EGTA or 45Ca2+ were followed by manual perfusion of a mixture of 300 μM ADP and 1 mM EGTA for 5 s. These measurements are represented by filled symbols in Figure 4:, Figure 5:. They confirm that after perfusion with EGTA or 45Ca2+, the phosphoenzyme was ADP-sensitive, as it was before perfusion, and that all the Ca2+ bound to the vesicles was bound to the ADP-sensitive phosphoenzyme, as expected from leaky vesicles.Figure 5:Ca2+ exchange from the phosphorylated ATPase (leaky vesicles). ATPase was first phosphorylated as in Fig. 3 and then perfused with 1 mM45Ca2+ plus 3 mM Mg2+ for various times (○, □). ADP sensitivity of the phosphoenzyme was evaluated by perfusing a mixture of 300 μM ADP and 1 mM EGTA (·, ■). ○, ·, Bound [45Ca]Ca2+; □, ■, phosphoenzyme. Cartoons illustrating the experiment: A, initial state; B, final state for 45Ca2+ (○, □).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The experiments reported in Figure 3:, Figure 4: were identical, except that the vesicles in Fig. 4 were leaky. Thus, although in the phosphorylated ATPase the Ca2+ sites are not accessible from the cytoplasm (Fig. 3), they are accessible from the lumen (Fig. 4). The use of leaky vesicles enabled EGTA (Fig. 4) and 45Ca2+ (Fig. 5) to induce Ca2+ dissociation, and moreover, 45Ca2+ impaired dephosphorylation. The stability of the ADP-sensitive phosphoenzyme during the perfusion with 45Ca2+ suggests that half the bound [45Ca]Ca2+ was replaced by 45Ca2+ on the Ca2E-P form of ATPase. This appears clearly in Fig. 6 which shows that the [45Ca]Ca2+/E-P ratio varies from 1.6 to 1 during the perfusion by 45Ca2+, whereas it remains around 1.8 during the dephosphorylation induced by EGTA. In the experiment reported in Figure 4:, Figure 5:, 3 mM Mg2+ were present, together with EGTA or 45Ca2+, in the perfusion buffers. Recalling that the phosphorylation step was done in the presence of 3 mM Mg2+versus 100 μM Ca2+, ATPase was phosphorylated by MgATP, so that we can assume the presence of Mg2+ at the catalytic site of the phosphoenzyme. Nevertheless, during the perfusion with 1 mM45Ca2+ and 3 mM Mg2+, partial substitution of 45Ca2+ for Mg2+ at the catalytic site cannot be excluded. ATPase turnover with CaATP as substrate is known to be much slower than with MgATP, possibly because of slower dissociation of Ca2+ from the Ca2E-P form when it has Ca2+ at its catalytic site(15Yamada S. Ikemoto N. J. Biol. Chem. 1980; 255: 3108-3119Abstract Full Text PDF PubMed Google Scholar). Such a substitution at the catalytic site during the perfusion could induce a slow phase in the Ca2+ dissociation kinetics that would be difficult to distinguish from a slow phase due to sequential dissociation from the transport sites. This possibility was tested by repeating the experiment described in Fig. 5 with various Mg2+ concentrations. Phosphorylated ATPase was formed as described in the legend to Fig. 5 and perfused with 1 mM45Ca2+ and either no added Mg2+ or 1, 3, or 5 mM Mg2+ (Fig. 7). The concentration of Mg2+ did not significantly modify the Ca2+ dissociation kinetics, as 45Ca2+ impaired dissociation of half the bound [45Ca]Ca2+ and dephosphorylation. The rate of the fast phase was 0.2-0.5 s-1, whereas the rate of the slow phase was the same as that of the phosphoenzyme, 0.02-0.03 s-1. If a Mg2+/Ca2+ exchange was to take place at the catalytic site during the perfusion, one would expect the amplitudes and the rates of the fast and slow phases to be modified by the Mg2+/Ca2+ ratio in the perfusion buffer. It is therefore more likely that the observed slow phase corresponds to a non-exchangeable Ca2+. Understanding how Ca2+ is transported from the cytoplasm to the SR lumen requires some knowledge of the Ca2+ binding sites on the different forms of ATPase. In the absence of ATP, when there is no turnover, the cytoplasmic Ca2+ binding reaction (step 4 in Fig. S1) has been well characterized, as it can be studied both kinetically and at equilibrium (1Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10953-10960Abstract Full Text PDF PubMed Google Scholar, 8Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10961-10968Abstract Full Text PDF PubMed Google Scholar, 18Ikemoto N. J. Biol. Chem. 1974; 249: 649-651Abstract Full Text PDF PubMed Google Scholar, 19Dupont Y. Biochem. Biophys. Res. Commun. 1976; 71: 544-550Crossref PubMed Scopus (97) Google Scholar, 20Inesi G. Kurzmack M. Coan C. Lewis D.E. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar, 21Guillain F. Gingold M.P. Bschlen S. Champeil P. J. Biol. Chem. 1980; 255: 2072-2076Abstract Full Text PDF PubMed Google Scholar). The situation is different for the Ca2+ binding sites on the phosphorylated ATPase (step 2 in Fig. S1), which should be studied during turnover. Knowledge of the lumenal affinity for Ca2+ can be obtained with tight vesicles loaded with Ca2+. For instance, the study of phosphoenzyme formation from Pi in the presence of various lumenal Ca2+ concentrations has yielded information on the lumenal affinity for Ca2+, which was found in the millimolar range at pH 7 and 20°C(22Prager R. Punzengruber C. Kolassa N. Winkler F. Suko J. Eur. J. Biochem. 1979; 97: 239-250Crossref PubMed Scopus (31) Google Scholar). Direct access to the Ca2+ binding sites on E-P requires working with leaky vesicles. In this case, as the affinity of the lumenal sites is lower than that of the cytoplasmic sites, addition of Ca2+ to E-P induces dephosphorylation and Ca2+ binding to E(23Guimaraes-Motta H. de Meis L. Arch. Biochem. Biophys. 1980; 203: 395-403Crossref PubMed Scopus (15) Google Scholar). Nevertheless, de Meis et al. (13de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar) have shown that working on leaky vesicles ATP could be synthesized in the absence of a Ca2+ gradient, provided that a mixture of Ca2+ and ADP was added to E-P. In this paper, ATP synthesis was studied as a function of the Ca2+ concentration under various conditions that yielded other estimations of the luminal affinity for Ca2+. Of particular interest is that at pH 8 and 0°C, half the maximal amount of ATP synthesized was obtained with 10 μM Ca2+, instead of 300 μM at pH 8 and 30°C or pH 7 and 0°C. Although these numbers should not be taken as absolute values for Ca2+ affinity, it is likely that alkaline pH and low temperature are conditions that increase the lumenal affinity for Ca2+. In the absence of any known possibility to measure Ca2+ binding to the phosphoenzyme directly, we have studied Ca2+ dissociation from phosphorylated ATPase toward the vesicle lumen using the rapid filtration technique. The experimental conditions were chosen to yield (i) as high as possible lumenal affinity for Ca2+, i.e. an affinity high enough to use reasonable concentrations of 45Ca2+ to compete with bound [45Ca]Ca2+, (ii) as much as possible Ca2+-bound phosphoenzyme, (iii) as low as possible ATPase activity, in order to have enough ATP in the 30-μl wet volume of the filter to maintain the ATPase phosphorylated during the few seconds necessary to start the rapid filtration experiment. All three requirements were achieved while working at pH 8, 5°C, 300 mM KCl and using 100 μM ATP for phosphorylation. Under these conditions, the ATPase activity was effectively low, and the vesicles were able to accumulate Ca2+ (Figure 1:, Figure 2:, Figure 3:) showing that the ATPase cycled normally. After phosphorylation, the phosphoenzyme was ADP-sensitive, and there were two Ca2+ bound per phosphoenzyme (Figure 3:, Figure 4:). Thus, the vast majority of ATPase was in the Ca2E-P form, in which Ca2+ is said to be occluded, because it cannot be released on the cytoplasmic side unless the ATPase has bound ADP. When this occluded Ca2+ was formed in leaky vesicles, perfusion with EGTA simultaneously induced dissociation of the two Ca2+ ions and dephosphorylation, as expected from the fact that hydrolysis of E-P, the Ca2+-deprived phosphoenzyme, is fast in the presence of KCl(14Shigekawa M. Akowitz A.A. J. Biol. Chem. 1979; 254: 4726-4730Abstract Full Text PDF PubMed Google Scholar). The luminal Ca2+ dissociation, i.e. the EGTA-induced dissociation of Ca2+ from Ca2E-P (Fig. 4) was slow compared with the cytoplasmic dissociation of Ca2+ from Ca2E (Fig. 2). It is likely that this slow rate illustrates Ca2+ deocclusion from the Ca2+-bound phosphoenzyme. Keeping in mind that hydrolysis of E-P is fast with KCl present and that the ADP sensitivity is lost together with Ca2+ dissociation, the fact that both the dephosphorylation and the Ca2+ dissociation are biphasic suggests that there is an equilibrium between two forms of Ca2E-P. One form, denoted as [Ca2]E-P, would bear occluded Ca2+, and the other form would be able to exchange Ca2+ with the lumen. Thus, biphasic dissociation of Ca2+ and biphasic dephosphorylation would be due to a fast dissociation of Ca2+ from the non-occluded form and the slow conversion of the occluded form into the non-occluded form. At this point, more details on the luminal dissociation of Ca2+ come from the Ca2+ exchange experiments. Comparison of the Ca2+ exchange experiments performed on the phosphorylated ATPase (Fig. 5) and on the nonphosphorylated ATPase (Fig. 2) shows that cytoplasmic Ca2+ exchange was faster than lumenal Ca2+ exchange. On the cytoplasmic side, this exchange is explained by the following sequence, where the departure of one 45Ca2+ is followed by the binding of one 45Ca2+. The same sequence on the phosphorylated ATPase suggests the existence of an intermediate phosphoenzyme having bound only one 45Ca2+ and able to bind one 45Ca2+. Taking into account the occluded state, Ca2+ exchange on the phosphorylated ATPase can be described by the following sequence. REACTION 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) Such a sequence would provide a stable phosphoenzyme in the presence of cold Ca2+. In the absence of lumenal Ca2+, sequential dissociation of both Ca2+ ions would thus occur following. Our data show a close similarity between the transport sites of the nonphosphorylated ATPase that are accessible from the cytoplasm and those of the phosphorylated ATPase that are accessible from the vesicle lumen. In both states, the transport sites interact with each other as bulk Ca2+ impairs the dissociation of one of the two Ca2+ bound. Our results agree with the conclusion of Inesi (5Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar) whose model includes luminal sequential dissociation, but they differ from those of Hanel and Jencks (9Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar) and Orlowski and Champeil (10Orlowski S. Champeil P. Biochemistry. 1991; 30: 11331-11342Crossref PubMed Scopus (37) Google Scholar) who found that the two Ca2+ ions could not be distinguished once the ATPase was phosphorylated. As stated above, this discrepancy is probably due to the difference in the experimental conditions. For instance, at room temperature the luminal affinity for Ca2+ is low and the ATPase activity is high, so that it is necessary to phosphorylate ATPase and to exchange cold Ca2+ for radioactive Ca2+ simultaneously, i.e. to perfuse at least 10 mM45Ca2+ and ATP together. In this case, an ATP/ADP-mediated Mg2+/Ca2+ exchange can occur in the filter, leading to various populations of phosphoenzymes displaying different rates at step 2 in Fig. S1, as discussed by Orlowski and Champeil (10Orlowski S. Champeil P. Biochemistry. 1991; 30: 11331-11342Crossref PubMed Scopus (37) Google Scholar). Under our conditions, there was only 1 mM45Ca2+ perfused, and ATP and ADP were washed out of the filter after the first 25 ms of perfusion; thus, there was very little possibility to have an ATP/ADP-mediated Mg2+/Ca2+ exchange at the catalytic site. There still remains the possibility that the effect of cold Ca2+ is due to a direct substitution of Ca2+ for Mg2+ at the catalytic site. Fig. 7 shows that this was not the case, because varying the Mg2+ concentration during the Ca2+ exchange did not change its kinetics. Finally, as Hanel and Jencks (9Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar) found the same rate of internalization of Ca2+ whether they monitored internalization of both ions or each individual ion into empty or Ca2+-loaded vesicles, they suggested that they could only measure a slow deocclusion rate preceding the Ca2+ dissociation steps. This slow deocclusion step was also seen in our experiments, but at variance with Hanel and Jencks(9Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar), we observed different effects when EGTA or 45Ca2+ was used. Thus, the comparison between their results and ours suggests that the experimental conditions modify the equilibrium between the occluded and deoccluded states of Ca2+ on the phosphoenzyme. Under our conditions, there would be enough Ca2+ in the deoccluded state to observe the dissociation of the first ion and thus the impairment of the dissociation of the second ion in the presence of cold Ca2+. Our finding that Ca2+ dissociation from the phosphorylated enzyme is sequential, as is Ca2+ binding to the nonphosphorylated enzyme, suggests at first sight that during the transport cycle, the Ca2+ ions cross the membrane sequentially via a channel-like structure such as the one sketched here. Nevertheless, a few points should be emphasized. First, a channel-like structure is obviously the simplest way to describe the interactions between the two Ca2+ sites, but it is not the only one. Second, a channel-like structure also suggests that the first ion bound to the cytoplasmic sites is the first ion to dissociate toward the lumen. Such a first-in-first-out model has been suggested by Inesi(5Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar). Third, there is a controversy about the possibility to have a channel-like structure crossing the membrane and including the putative Ca2+ sites. Site-directed mutagenesis has yielded information on the location of the Ca2+ sites, as six charged residues from the membrane helices M4, M5, M6, and M8 (Glu309, Glu771, Asn796, Thr799, Asp800, and Glu908) were shown to be crucial for Ca2+ transport(24Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (470) Google Scholar), and among them, the five residues belonging to M4, M5, and M6 were shown to be crucial for Ca2+ occlusion(25Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 25739-25743Abstract Full Text PDF PubMed Google Scholar, 26Andersen J.P. Vilsen B. J. Biol. Chem. 1994; 269: 15931-15936Abstract Full Text PDF PubMed Google Scholar). According to Inesi (27Inesi G. Biophys. J. 1994; 66: 554-560Abstract Full Text PDF PubMed Scopus (22) Google Scholar), M4, M5, M6, and M8 could form a channel providing the residues coordinating the two Ca2+ ions at its inner surface. According to Andersen(28Andersen J.P. Vilsen B. FEBS Lett. 1995; 359: 101-106Crossref PubMed Scopus (114) Google Scholar), the three residues belonging to M6 cannot at the same time coordinate the two Ca2+ ions and be part of the same α-helix. We have shown here that the lumenal dissociation of the Ca2+ ions is compatible with a channel-like structure, because it is sequential. However, we did not try to obtain any information about what happens in the occluded state, as we did not follow a specified Ca2+ ion, i.e. the first or the second, from the cytoplasmic side to the lumenal side." @default.
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