©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Variable Stoichiometric Efficiency of Ca and Sr Transport by the Sarcoplasmic Reticulum ATPase (*)

(Received for publication, November 7, 1994)

Xiang Yu Giuseppe Inesi

From the Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In comparative experiments with Ca ATPase in native sarcoplasmic reticulum vesicles and reconstituted proteoliposomes, we find that a variable stoichiometry of Ca or Sr transport per ATPase cycle is observed in the absence of passive leak through independent channels. The observed ratio is commonly lower than the optimal value of 2 and depends on the composition of the reaction mixture. In all cases, a progressive rise in the lumenal concentration of Ca and Sr is accompanied by a parallel reduction of coupling ratios. Significant ATPase activity remains even after asymptotic levels of Ca accumulation are reached. This residual activity subsides if the Ca concentration in the outer medium is reduced below activating levels (as it would following Ca transients in muscle fibers). The reduction of stoichiometric coupling is explained with a reaction scheme, including a branched pathway for hydrolytic cleavage of phosphorylated intermediate before release of Ca into the lumen of the vesicles. Flux through this pathway is favored when net lumenal Ca dissociation from the phosphoenzyme is impeded and results in P(i) production accompanied by lumenal and medium Ca exchange. Occurrence of reactions through branched pathways may have general implications for the stoichiometric efficiency of energy-transducing enzymes.


INTRODUCTION

Vesicular fragments of sarcoplasmic reticulum (SR) (^1)membrane provide a convenient experimental system for studies of active Ca transport coupled to ATP utilization. Early experiments with this system (Hasselbach, 1964) indicate that two Ca are transported into the lumen of the vesicles in parallel with utilization of one ATP. It was later demonstrated that each ATPase requires cooperative binding of two Ca for catalytic activation (Meissner, 1973; Inesi et al., 1980). Therefore, transport of 2 Ca/catalytic cycle occurs if both bound Ca are translocated and is equivalent to maximal stoichiometric efficiency of the pump.

Ratios of 2 (or nearly 2) Ca/ATP utilized have been observed under conditions permitting free Ca to remain low in the lumen of the vesicles: (a) in steady state experiments in which oxalate is used for complexation of lumenal Ca (Martonosi and Feretos, 1964) and (b) in pre-steady state experiments in which lumenal Ca has yet to rise (Inesi et al., 1978). On the other hand, Ca/ATP ratios lower than 2 are commonly observed under other conditions permitting lumenal Ca to rise. Such lower ratios have been generally attributed to leak of transported Ca through pathways other than the ATPase. In fact, most diagrams represent the ATPase cycle with a pathway of sequential reactions which, in principle, do not allow uncoupling of Ca transport and ATP utilization (Fig. 1, solid line). On the other hand, a variable stoichiometry of active transport may be considered to be an intrinsic feature of the pump if the reaction scheme includes alternate pathways leading to hydrolytic cleavage of P(i) without vectorial displacement of Ca (Johnson et al., 1985; Berman and King, 1990). We have now obtained experimental evidence for this latter alternative, using native SR and reconstituted vesicles to measure coupling ratios in the presence of gradually rising lumenal Ca (or Sr) and in the virtual absence of passive leak. We find that the optimal 2:1 ratio is reduced in proportion to the lumenal concentration of transported cation. The uncoupling is accounted for by a branched pathway (Fig. 1, dotted line) of the ATPase cycle, permitting cleavage of the phosphorylated intermediate before net release of Ca into the lumen of the vesicles.


Figure 1: A simple diagram of the Ca ATPase catalytic cycle. Only four measurable reactions are shown here: Ca binding to the enzyme on the outer surface of the vesicles in exchange for H, enzyme phosphorylation by ATP, dissociation of bound Ca in the lumen of the vesicles, and hydrolytic cleavage of the phosphorylated enzyme intermediate. Isomeric transitions of these intermediates (such as E1 to E2, and E-P1 to E-P2) are required to explain vectorial translocation of bound Ca (De Meis and Vianna, 1979) and the kinetic behavior of the enzyme in transient state (Froelich and Taylor, 1975; Inesi et al., 1980; Petithory and Jencks, 1986).




MATERIALS AND METHODS

Octaethylene glycol-n-dodecyl ether (CE(8)) was obtained from Nikko Chemical Co (Tokyo). Purified egg yolk phosphatidylcholine and phosphatidic acid were from Avanti Polar Lipid Inc., Ca and [-P]ATP were from DuPont NEN, n-octyl-beta-D-glucopyranoside, calcimycin (A23187), and all other materials were obtained from Sigma.

SR vesicles were prepared from rabbit hind leg muscle by the method described by Eletr and Inesi(1972). This method selects vesicles derived from longitudinal SR membrane and containing negligible amounts of the ryanodine receptor Ca channel associated with junctional SR. The protein concentration was measured by the method of Lowry et al.(1951), standardized with bovine serum albumin.

Preparation of unilamellar liposomes by reverse phase evaporation and reconstitution of Ca transport ATPase with liposomes were carried out as described previously (Yu et al., 1994). The size, the ATPase orientation, and the low permeability of these proteoliposomes were recently characterized in detail (Levy et al., 1992).

Ca uptake by native SR vesicles was followed by determining the association of radioactive Ca tracer with the vesicles after elimination of the reaction medium by filtration through Millipore 0.45-µm HWP nitrocellulose membrane. The reaction mixture contained 10 mM PIPES, pH 7.0, 100 mM KCl, 5 mM MgSO(4), 0.2 mM (Ca)CaCl(2), 0.2 mM EGTA, and 20 µg of SR protein/ml. The reaction was started by the addition of 0.1 mM ATP.

Measurements of lumenal and medium Ca exchange were obtained by first loading the SR vesicles with Ca as above for 3 min and then adding 4 ml of reaction medium containing non-radioactive Ca to 1 ml of reaction mixture. The reaction was stopped at serial times by addition of 3 ml of LaCl(3) solution (4 mM LaCl(3), 10 mM PIPES, pH 7.0). The quenched samples were then filtered through Millipore membranes and washed with 10 ml of LaCl(3), 10 mM PIPES, pH 7.0. The radioactivity was determined by scintillation counting.

Ca or Sr uptake by the proteoliposomes was measured by continuously monitoring differential (750/650 nm) absorption changes undergone by the metallochromic indicator Arsenazo III (Scarpa, 1979). The reaction was carried out in the cuvette of an SLM-Aminco double wavelength spectrophotometer. In most cases the reaction medium contained 10 mM PIPES, pH 7.0, 100 mM KSCN (or K(2)SO(4)), 5 mM MgSO(4), 50 µM Arsenazo III, and 0.8 mg of proteoliposomal lipid/ml. CaCl(2) or SrCl(2) was added to the cuvette after obtaining the absorption base line, and the reaction was started by addition of 0.1 mM ATP. The output from the spectrophotometer was recorded directly or acquired with a Nicolet digital oscilloscope.

ATP hydrolysis in native vesicles was followed by measuring production of P(i) by the calorimetric method described by Lanzetta et al.(1979). The reaction mixture contained 10 mM PIPES, pH 7.0, 100 mM KCl, 5 mM MgSO(4), 0.2 mM CaCl(2), 0.2 mM EGTA, and 20 µg of SR protein/ml. The reaction was started by the addition of 0.1 mM ATP and quenched at serial times by mixing an aliquot with the color reagent.

ATP hydrolysis by reconstituted proteoliposomes was measured by enzyme coupled assay according to the method described by Horgon et al.(1972). The absorption change at 340 nm was continuously monitored in a mixture containing 10 mM PIPES, pH 7.2, 100 mM KCl, 5 mM MgSO(4), 20 µM CaCl(2), 400 µg of proteoliposomal lipid/ml, 25 units of pyruvate kinase/ml, 25 units of lactic dehydrogenase/ml, 2 mM phosho(enol)pyruvate, and 150 mM NADH. The reaction was started by addition of 0.1 mM of ATP.

Phosphorylated intermediate formation by the Ca ATPase of native SR vesicles was determined by measuring the incorporation of [-P]ATP terminal phosphate into the protein. The reaction mixture contained 10 mM PIPES, pH 7.0, 100 mM KCl, 5 mM MgSO(4), 0.2 mM EGTA, 0.2 mM CaCl(2), and 20 µg of SR protein/ml. The reaction was started by the addition of 0.1 mM [-P]ATP and quenched at serial time by mixing aliquots of the mixture with equal volumes of 1 M perchloric acid and 4 mM NaH(2)PO(4). The denatured protein was collected on 0.45-µm Millipore filters and washed with 30 ml of 0.125 M perchloric acid and 2 mM NaH(2)PO(4). The filters were then dissolved in N,N-dimethylformamide and the radioactivity determined by scintillation counting.


RESULTS

Coupling Ratios with Native SR Vesicles

When native SR vesicles are incubated with ATP and oxalate in the absence of Ca (i.e. excess EGTA present) a very low rate of ``Ca-independent ATPase'' is observed. Upon addition of Ca, the rate of ATP hydrolysis rises in parallel with Ca accumulation by the vesicles (Hasselbach, 1964). In this type of experiment, the oxalate added to the reaction mixture precipitates Ca as it rises in the lumen of the vesicles and limits the free lumenal Ca concentration to the calcium oxalate dissociation constant (<0.1 mM). Under these conditions then, the rates of Ca uptake and Ca-dependent ATP utilization remain constant (Fig. 2), maintaining a stoichiometric ratio of approximately 1.5. Therefore, the stoichiometric efficiency of the pump is nearly maximal and remains constant for several minutes.


Figure 2: Ca uptake and ATP hydrolysis by native SR vesicles in the presence of oxalate. Reaction mixture: 20 µg of SR protein/ml, 10 mM PIPES, pH 7.0, 100 mM KCl, 5 mM MgSO(4), 0.2 mM (Ca)CaCl(2), 0.2 mM EGTA, and 5 mM oxalate. The reaction was started by the addition of 0.1 mM ATP. 25 °C. The P(i) release shown is the difference between P(i) release in the presence and in the absence of Ca.



If similar experiments are repeated in the absence of oxalate, the ATP-dependent Ca accumulation soon reaches an asymptotic level, as the concentration of lumenal Ca rises rapidly (Fig. 3A). The ATPase activity, however, is not inhibited in parallel with accumulation of Ca. Therefore, the Ca:ATP ratio is reduced within a few seconds after the addition of ATP. This phenomenon can be observed with a better time resolution at low temperature (Fig. 3B), due to the lower rates of catalytic and transport activity. The initial Ca:ATP ratio of nearly two demonstrates that the native functional state of the enzyme is retained in these preparations.


Figure 3: Ca uptake and ATP hydrolysis by native SR vesicles in the absence of oxalate. Reaction mixture: 20 µg of SR protein/ml, 10 mM PIPES, pH 7.0, 100 mM KCl, 5 mM MgSO(4), 0.2 mM CaCl(2), and 0.2 mM EGTA. The reaction was started by the addition of 0.1 mM ATP. A, 25 °C; 1.0 mM EGTA was added where indicated. B, 10 °C.



It is noteworthy that the decline in Ca accumulation is not due to passive leak through collateral pathways since, if the ATPase activity is stopped by addition of the highly specific inhibitor thapsigargin (Sagara and Inesi, 1991), no significant leak of the accumulated Ca is observed (Fig. 4). Notwithstanding the lack of net loss of Ca load, lumenal and medium Ca undergo rapid exchange which is also prevented by thapsigargin (Fig. 5, see also Takakuwa and Kanazawa (1981) and Soler et al.(1990)). It should be pointed out that very little ADP-ATP or P(i)-ATP exchange (i.e. energy conserving reversal of the cycle through the solid line in Fig. 1, see also Ebashi and Lippman(1962); De Meis and Carvalho, 1974) occurs under our experimental conditions, owing to the very low ADP and P(i) concentrations. On the contrary, Ca exchange is accompanied by net P(i) production.


Figure 4: Ca accumulated by native SR vesicles in the absence of oxalate is not released following addition of thapsigargin (TG). Ca uptake was obtained as described for Fig. 2at 25 °C. Thapsigargin was added where 30 s following ATP, to reach a 1.0 µM concentration which inhibits the ATPase completely.




Figure 5: Ca exchange by loaded native SR vesicles. Reaction mixture: 20 µg of SR protein/ml, 10 mM PIPES, pH 7.0, 100 mM KCl, 5 mM MgSO(4), 0.2 mM (Ca)CaCl(2), 0.2 mM EGTA. The reaction was started by the addition of 0.1 mM ATP. 4 volumes of reaction medium with non-radioactive Ca was added at 3 min; 25 °C.



The comparative experiments performed in the presence and in the absence of oxalate indicate that the stoichiometric ratio of Ca transport and ATP utilization is reduced by the Ca concentration rise in the lumen of the vesicles. Furthermore, they indicate that high lumenal Ca favors hydrolytic cleavage of intermediate through an alternate pathway which results in production of P(i) and exchange of lumenal and medium Ca, but no net release of Ca into the lumen of the vesicles (pathway indicated by a dotted line in Fig. 1).

Cleavage of Phosphoenzyme at Low or High Lumenal Ca

If only one of two branched pathways of intermediate (E-PbulletCa(2) in Fig. 1) cleavage is inhibited by lumenal Ca, and the rate constant of the alternate pathway is lower than that of the productive pathway, the overall velocity of P(i) production is expected to decrease as the lumenal Ca rises, as in fact is shown in Fig. 3. Furthermore, the steady state level of phosphorylated enzyme intermediate is expected to be higher as a consequence of lumenal Ca rise. This can be tested in experiments carried out in the absence or in the presence of an ionophore which prevents net Ca accumulation by facilitating leak. It is shown in Fig. 6that, following addition of ATP to SR vesicles preincubated with Ca, the phosphoenzyme intermediate rises rapidly and then settles to a steady state level which is higher for the vesicles sustaining net Ca uptake than for vesicles unable to accumulate Ca.


Figure 6: Phosphoenzyme levels in SR vesicles with high or low lumenal Ca. Reaction mixture: 20 µg of SR protein/ml, 10 mM PIPES, pH 7.0, 100 mM KCl, 5 mM MgSO(4), 0.2 mM MgCl(2), and 0.2 mM EGTA in the presence or absence of 5 µM A23187. The reaction was started by the addition of 0.1 mM [-P]ATP. Incubation in ice.



Coupling Ratios with Reconstituted Proteoliposomes

A shortcoming of experiments performed with native SR vesicle is due to the high density of ATPase units within the membrane plane and the consequently low ratio of lumenal volume per ATPase unit. For this reason a high (mM) lumenal Ca concentration is reached with a few ATPase turnovers, and further uptake is precluded unless lumenal Ca is clamped with oxalate. We have then turned to a reconstituted proteoliposomal system (Levy et al., 1992) in which only four or five ATPase units are incorporated per vesicle. In this system, due to a favorable ratio of lumenal volume per ATPase unit, the lumenal Ca concentration rises very slowly and the Ca/ATP coupling ratio can be monitored during such a slow rise. Although the initial coupling ratio is slightly lower than observed with native vesicles, it is possible to demonstrate clearly that the ratio decreases gradually as lumenal Ca rises (Fig. 7A). On the other hand, the residual ATPase activity remains high even after the asymptotic level of Ca uptake is reached. If EGTA is added to the medium at this time, the residual ATPase activity ceases, indicating that it is still dependent on medium (outside the vesicles) Ca for activation. Most importantly, if the residual ATPase activity is stopped by the addition of thapsigargin, no significant leak of accumulated Ca is observed (Fig. 7A). Therefore, the observed reduction of coupling ratio is not due to passive leak of Ca, but is rather a feature of the ATPase mechanism itself.


Figure 7: Ca (A) or Sr (B) uptake and ATPase activity sustained by reconstituted proteoliposomes. Reaction mixture: 0.4 mg of proteoliposomal lipid/ml, 10 mM PIPES, pH 7.0, 100 mM KSCN, 5 mM MgSO(4), and 20 µM Ca or 100 µM Sr. The mixture for Ca uptake contained Arsenazo III, and that for ATPase containing a coupled detection system as described under ``Materials and Methods.'' The reaction was started by the addition of 0.1 mM ATP. Thapsigargin was added 10 min following the addition of ATP to reach a 1.0 µM concentration. 25 °C.



Comparative Experiments with Sr

To check further whether affinity and saturation of the phosphoenzyme divalent cation sites is involved in limiting the asymptotic level of Ca uptake and the stoichiometric ratios of Ca uptake and ATPase activity, we turned to Sr. It is known that Sr is transported by the SR ATPase with a mechanism analogous to the one used for Ca (Berman and King, 1990; Fujimori and Jencks, 1992a, 1992b; Orlowski and Champeil, 1993), but with a lower affinity of the binding sites on the enzyme before and after phosphorylation. We found that the maximal levels of Sr accumulation by the proteoliposomes are higher than the levels of Ca accumulation (Fig. 7B), likely due to the higher concentration of lumenal Sr required to saturate the ATPase cation binding sites in their low affinity and inward oriented state (Guimaraes-Motta et al., 1984). Here again, the Sr/ATP coupling ratio decreases as the lumenal Sr rises. Most interestingly, however, we found that favorable Sr/ATP ratios are maintained longer than the Ca/ATP ratios in analogous experiments (compare Fig. 5and Fig. 7). It should be pointed out that, in analogy to the experiments with Ca, we did not observe any significant leak of accumulated Sr when we stopped the ATPase pump with thapsigargin (Fig. 7B).

Transport inhibition by lumenal Ca or Sr can be attributed to saturation of the phosphoenzyme Ca binding sites in their state of low affinity and inward orientation, with consequent accumulation of loaded intermediate (E-PCa(2) in Fig. 1). It is of interest in this regard that acid pH lowers, and alkaline pH increases the affinity of the Ca sites in both the outward and inward orientations (Verjovski-Almeida and De Meis, 1977). In this connection, we reported previously (Yu et al., 1993) that higher levels of Ca accumulation are obtained when isothiocyanate, rather than chloride or sulfate, is the prevalent anion, due to the ability of isothiocyanate to dissociate H and compensate for lumenal H loss consequent to H/Ca countertransport. We then performed experiments to check whether replacement of isothiocyanate with sulfate would influence net Sr uptake and Sr/ATP stoichiometry by affecting the affinity of the inward oriented Ca sites. In fact, we found that when sulfate rather than isothiocyanate was used, both the asymptotic level of Sr uptake and the Sr/ATP ratio were reduced (Fig. 8).


Figure 8: Sr uptake by reconstituted proteoliposomes in the presence of different anions. Reaction mixture: 0.4 mg of proteoliposomal lipid/ml, 10 mM PIPES, pH 7.0, 100 mM K(2)SO(4) (A) or KSCN (B), 5 mM MgSO(4), and 100 µM Sr. The reaction were started by the addition of 0.1 mM ATP; 25 °C.



The comparative experiments on Sr and Ca uptake, as well as on the effects of sulfate and isothiocyanate, indicate that inhibition of net Ca uptake and reduction of stoichiometric efficiency are both related to the extent of divalent cation binding to the enzyme sites exposed to the lumen of the vesicles. In this context, the stoichiometric efficiency of the pump is a kinetically controlled variable which decreases progressively as the lumenal concentration of transported cation rises. It should be pointed out that in no instance we found the Ca/ATP ratio to exceed the value of two, which is the maximal ratio permitted by the stoichiometry of enzyme sites.


DISCUSSION

The idea of uncoupling in energy transducing enzymes derives from the effect of ionophores such as paranitrophenol and carbonyl cyanide p-trifluoromethoxyphenylhydrazone which facilitate leak of H across mitochondrial membranes, thereby collapsing the electrochemical gradient and uncoupling electron transport from ATP synthesis (Mitchell, 1979; Penefsky and Cross, 1991). An analogous effect is produced by ionophores such as X-537A or A23187 which produce leak of Ca across SR membranes, thereby preventing formation of transmembrane Ca gradients and uncoupling ATP utilization from net Ca accumulation (Scarpa et al., 1972). On the other hand, we find that the stoichiometric ratio of Ca uptake and ATPase activity can be also reduced in the absence of passive Ca leak. Therefore, the phenomenon observed in these experiments is inherent to the ATPase mechanism itself. We also find that, given an initial coupling ratio for native SR or proteoliposomal vesicles (which may be influenced by the reaction mixture and/or the enzyme preparation), such a ratio is reduced in parallel with rise of lumenal Ca (or Sr) and saturation of the lumenal phosphoenzyme sites with the divalent cation. While net Ca uptake is reduced by the rise of lumenal Ca, the ATPase activity is inhibited most effectively by a reduction of the Ca concentration in the outer medium rather than by the rise of lumenal Ca.

It is apparent from the reaction scheme in Fig. 1(solid lines only) that a rise of lumenal Ca ([Ca]) would interfere with progress of the E-PbulletCa(2) intermediate through the cycle, thereby producing inhibition of both Ca uptake and ATPase activity with no change of their coupling ratio. Therefore, in order to explain the observed uncoupling effect of lumenal Ca in the absence of a Ca leak, it is necessary to postulate an alternate pathway for hydrolytic cleavage of the intermediate before release of bound Ca into the lumen of the vesicles (dotted lines in Fig. 1). The occurrence of lumenal and medium Ca exchange, in the absence of energy conserving ATP-ADP and P(i)-ATP exchanges, is consistent with significant flux through this alternate pathway (dotted line in Fig. 1). Furthermore, the increased steady state level of phosphorylated intermediate observed in the presence of high lumenal Ca suggests that the rate constant of the alternate pathway is lower than that for the primary pathway.

We tested these intuitive predictions by adding an alternate pathway to a reaction sequence previously utilized to calculate steady state fluxes and levels of intermediates, based on the bidirectional rate constants of the partial reactions and the concentrations of substrates and products. The reaction sequence (Fig. 9) includes second order association and dissociation events, as well as isomeric transitions as required to explain the vectorial changes of orientation, and the kinetic and cooperative behavior of the enzyme under different conditions (Inesi et al., 1980; Fernandez-Belda et al., 1984). With specific reference to the branched pathway, we can see in the diagram (Fig. 9) that the intermediate E`-PCaCa, formed by phosphorylation of E`CaCa with ATP and release of ADP (reactions G and H), undergoes an isomeric transition (reaction I) to a state (*E`-PCaCa) from which the bound Ca can be released into the lumen of the vesicles. *E`-PCaCa then undergoes hydrolytic cleavage (reactions J to N) thereby completing a productive cycle. Alternatively, E`-PCaCa can undergo hydrolysis before releasing Ca into the lumen of the vesicles, thereby returning to the E`CaCa state (reactions F and E) from which the bound Ca is released to the medium outside the vesicles (reactions C to A) at the end of an unproductive cycle.


Figure 9: Diagram of a reaction scheme used for computation of steady state velocities and intermediate states. Bidirectional rate constants (s) of each sequential step and the concentrations of substrates and products used in the computations are given in the diagram. A detailed description of this analysis and computation method is given in Inesi et al. (1988).



Computations based on this scheme make it clear that a rise in lumenal Ca favors flux through the alternate pathway which allows the intermediate to undergo hydrolytic cleavage without releasing Ca into the lumen of the vesicles. Consequently, the overall coupling ratio is reduced when lumenal Ca rises ( Fig. 3and Table 1), as observed experimentally. An additional finding predicted by the computations is a significant increase of the steady state level of phosphorylated intermediate in the presence of high lumenal Ca ( Table 1and Fig. 6). It is also predicted by such computations (not shown) that, given the same concentration of divalent cation in the lumen of the vesicles, the flux through the branched pathway is affected by the affinity of the phosphoenzyme for the divalent cation and by the kinetic constants of each pathway. This explains the more favorable coupling ratios observed in our experiments when Sr is used instead of Ca, since the affinity of the phosphoenzyme for Sr is lower than the affinity for Ca. It also explains the more favorable ratios observed when the lumen of the vesicles is maintained acid by replacing sulfate with isothiocyanate (Fig. 7), since the affinity of the phosphoenzyme for Ca is lower at acid pH.



Our experiments raise a general question of whether it is reasonable to expect fixed coupling ratios in energy transducing enzymes, as predicted by schemes outlining rigid sequences of partial reactions. In principle, a protein equilibrated with various ligands will yield a statistical distribution of all possible species and isomers, depending on the appropriate binding and isomerization constants (Hill, 1991). In the case of an enzyme, each species should contribute to the overall reaction flux depending on its appropriate kinetic constant and its steady state concentration. In this paper we have considered only the simple case of one alternative pathway, assuming that all other possible branched reactions occur at negligible rates. This simple analysis is sufficient to explain the experimentally observed behavior and to underline a general point of interest regarding the coupling stoichiometry of energy transducing enzymes.

A final point of interest is related to the physiology of cytosolic Ca removal by the transport ATPase associated with intracellular organelles. Our present studies suggest that following a cytosolic Ca transient, such as that involved in contractile activation of muscle fibers, the transport ATPase remains active until enough Ca is removed to reach a cytosolic concentration below the level required for activation of the Ca ATPase. This mechanism would insure relaxation of other Ca-dependent cytosolic processes, such as contraction of myofilaments. The calcium capacity of the organelles would then be determined by the affinity of the enzyme lumenal sites for Ca and by the presence of Ca-binding proteins within the organelles.


FOOTNOTES

*
This research was supported by Grant HL27867-13 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: SR, sarcoplasmic reticulum; PIPES, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

We are grateful to Lilin Zhong (rotating student) for her participation in some of the experiments.


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