Sequential Reactions in Pi Utilization for ATP Synthesis by Sarcoplasmic Reticulum*

THE JOURNAL OF BI~L~CICAL CHEMISTRY Vol 54. No. 19. Issue of October 10, pp. 9464-9468. 1979 Prmted,n L.S.A. Sequential Reactions in Pi Utilization for ATP Synthesis by Sarcoplasmic Reticulum* (Received for publication, March 26, 1979) Ricardo M. Chaloub, Horatio Guimaraes-Motta, Sergio Verjovski-Almeida, and Leopold0 de Meis From the Departamento de Bioquimica, Centro de Ciencias da Saude, Universidade Federal, Rio de Janeiro, 21910 Brazil Giuseppe Inesi From the Laboratory of Physiology and Biophysics, University of the Pacific, San Francisco, California 94115 Incorporation of Pi and ATP synthesis by sarcoplasmic reticulum ATPase were studied by rapid quench methods in order to demonstrate whether protein phosphorylation obtained in various experimental conditions leads to intermediates of the same reaction chain, or to products of independent reactions. Phosphorylation occurs rapidly (k = 30 s- ) when saturating Pi is added to vesicles preincubated with ethylene glycol bis(p-aminoethyl ether)iv,iv,iv,iv -tetraacetic acid (EGTA), while it is much slower (k = 2.0 s- ) in vesicles loaded actively (with calcium in the presence of ATP) previous to sequential addition of EGTA and Pi. If the reaction is started by simultaneous rather than sequential addition of EGTA and Pi, a lag period is observed and phosphorylation occurs with identical kinetics in loaded and nonloaded vesicles. In all cases, independent of the different kinetics of ATPase phosphorylation with Pi, decay of the resulting phosphoenzyme occurs with identical kinetics upon Pi dilution, consistent with hydrolytic cleavage of the same species. The rate constant for the hydrolytic reaction (8 to 11 s-l) is equal to the turnover of the enzyme operating with ATP as the substrate. Addition of Pi and ADP to loaded vesicles, independent of whether EGTA is added previous to or simultaneous with Pi, results in ATP synthesis with kinetics similar to those of Pi incorporation into the enzyme. It is concluded that ATPase phosphorylation with Pi occurs exclusively when the enzyme resides in specific reactive states which are part of the same catalytic cycle. Observed differences in the kinetics of phosphorylation are related to different states in which the enzyme resides in various experimental conditions and reflect conversion of the enzyme to a reactive state. Independent of the kinetics of ATPase phosphorylation with Pi, the resulting phosphoenzyme acquires the ability to synthesize ATP in the presence of high intravesicular Ca +. The catalytic and transport cycle of SR ATPase includes * This work was supported by the Conselho National de Desenvolvimento Cientifico e Tecnologico (CNPq), Universidade Federal de Rio de Janeiro, and Financiadora de Estudos e Projetos of Brazil, and by United States Public Health Service Grant HL 16607. Participation (of G. I.) in this work was rendered possible through a visiting scientist program of the CNPq. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 USC. Section 1734 solely to indicate this fact. The abbreviations used are: SR, sarcoplasmic reticulum; EGTA, ethylene glycol bis(p-aminoethyl ether)n,n,n,n -tetraacetic acid. a phosphorylated intermediate formed by incorporation of ATP terminal phosphate into the enzyme (l-4). Enzyme phosphorylation by ATP is dependent on Ca binding to high affinity sites located on the outer surface of the SR vesicles and is rapidly followed by Ca + translocation across the membrane and a marked reduction of the Ca binding association constant (5,6). Thereby, Ca2+ is released against a concentration gradient, and the phosphoenzyme undergoes hydrolytic cleavage. Conversely, in the presence of a Ca concentration gradient, addition of P, and ADP cause reversal of the pump, leading to Ca + efflux and ATP synthesis (7, 8). Reversal of the cycle includes a phosphorylated intermediate formed by enzyme incorporation of Pi which is then transferred to ADP (9-11). An important finding related to the mechanism of Pi utilization is that phosphorylation of intact or leaky vesicles with Pi can be obtained even in the absence of a transmembrane Ca2+ gradient (12-17). In these conditions, the phosphoenzyme cannot transfer its phosphate to ADP (17,18). However, upon addition of high (>mm) Ca*, a rapid Pi ++ ATP exchange is observed (19-21) and, in some conditions, a small amount of net ATP synthesis is detected (15-22). Presently, owing to differences in kinetic features and in the ability to form ATP, it is uncertain whether the phosphoenzymes formed in the presence or in the absence of a transmembrane Ca*+ gradient are intermediates of the same reaction chain or, rather, products of reactions proceeding in parallel or branched chains (23). In this report, evidence is presented, indicating that ATPase phosphorylation with Pi in the presence or in the absence of a transmembrane Ca + gradient, leads to the same intermediate which acquires the ability to synthetize ATP upon Ca* binding to low affinity sites facing the inner surface of the vesicles. Distinctive kinetic features of Pi incorporation and cleavage correspond to discrete sequential steps of an ordered reaction sequence. EXPERIMENTAL PROCEDURES Sarcoplasmic reticulum vesicles were prepared from rabbit skeletal muscle as previously described (24). P% was obtained from the Brazilian Institute of Atomic Energy and was purified by extraction as phosphomolybdate with isobutyl alcohol/benzene, re-extracted to the aqueous phase with ammonium hydroxide solution, and finally precipitated as the MgNH4P04 salt (12). The P, was stored in dilute HCl until used. Loading of the Vesicles with Calcium Phosphate-The SR vesicles were actively loaded with calcium phosphate when indicated, by incubation at room temperature for 5 min in a medium containing 20 mm Tris/maleate (ph 6.5), 5 mm MgClz, 1 mm %!aclz, 0.7 mm EGTA, 20 mm P,, 2 mm ATP, and 0.65 mg of SR protein/ml. The mixture was subsequently distributed into eight tubes and centrifuged at 40,000 x g for 30 min at 4 C. The supernatants were poured and 9464

ATPase Phosphorylation and ATP Synthesis 9465 aliquots were counted to estimate the amount of calcium taken up by the vesicles. The pellets were kept ice cold. The vesicles were resuspended a few seconds before utilization in different media as described below. The SR suspensions thus obtained were used within 5 min. The Ca*+ loading soon after centrifugation was 1.07 f 0.05 pmol of Ca +/mg of protein and decreased to 0.57 -C 0.10 pmol of Ca /mg of protein after 5 min in the resuspension media at 25-28 C. Enzyme Preincubation with EGTA-In the experiments where the enzyme was exposed to EGTA previous to phosphorylation, the vesicles (either nonloaded or Ca* -loaded vesicles) were suspended in a medium containing 20 mm Tris/maleate (ph 6.2), 10 mm MgC12, 2 mm EGTA, and 1.2 mg of SR protein/ml. To measure the time course of phosphorylation, the SR suspension was placed in Syringe A of the rapid mixer and the reaction was started by adding an equal volume of Solution B: 20 mm Tris/maleate (ph 6.2), 10 mm MgCla, 2 mm EGTA, and 0.4 to 4.0 mm 32P1. To measure dephosphorylation, the SR suspension was hand-mixed with an equal volume of Mixture B and the resulting phosphoenzyme suspension was immediately placed in the rapid mixer. Dephosphorylation was started by two means: either by a lo-fold dilution of the P, or by inhibiting phosphorylation with the addition of a CaZ - containing solution. The reactions were quenched with 250 mm HClO, plus 4 mm Pi carrier. The denatured phosphoprotein (0.6 to 1 mg) was washed four times by centrifugation and resuspension in ice cold 125 mm HClOl plus 2 mm P, carrier. The washed pellet was dissolved in 0.25 ml of a solution containing 0.2 M Na&O:s, 0.1 N NaOH, 70 mm sodium dodecyl sulfate and 5 mm P,. An aliquot was taken for micro determination of protein concentration (25) and liquid scintillation counting of P. Alternatively, the denatured phosphoprotein (0.1 to 0.2 mg) was washed by depositing a known volume of quenched suspension onto a glass-fiber filter and flushing with acid as previously described (26). The filter was counted in liquid scintillation fluid. Enzyme Preincubation with Ca +-When indicated, SR vesicles (nonloaded or Ca +-loaded vesicles) were incubated with Ca + previous to phosphorylation. This was done by suspending the vesicles in a medium from which EGTA was omitted and containing 20 mm Tris/maleate (ph 6.2), 10 mm MgC12, 50 PM CaC12, and 1.2 mg of SR protein/ml. Phosphorylation and dephosphorylation were started and stopped as indicated above. The EGTA concentration in Medium B was increased to 4 mm. AZ P Synthesis-Loaded vesicles were suspended in 20 mm Tris/ maleate (ph 6.2), 10 inm MgC12, and either 5 mm EGTA or 50 pm CaCIP. Phosphorylation was started in the rapid mixer by adding an equal volume of 20 mm Tris/maleate (ph 6.2), 10 mm MgC12, 4 mm 12P,, 10 mm glucose, 0.2 mm ADP, 5 units/ml of hexokinase, and either 5 or 10 mm EGTA. The reaction was quenched with 250 mm HClO, plus 4 mm P, carrier. After quenching, ATP was added to a final concentration of 2 mm to carry the radioactive ATP synthesized which had not been trapped by hexokinase. The denatured protein was pelleted and washed as described above. The supernatant (3 ml) was mixed with 0.4 ml of concentrated HCl, 0.75 ml of 60 mm ammonium molybdate in 0.01 N HCl, and 0.6 ml of acetone, and vigorously blended on a Vortex mixer for 40 s with 8 ml of isobutyl alcohol/benzene (l:l, v/v). The organic solvents in the upper phase were then discarded, 0.02 ml of 20 mm P, carrier and 0.6 ml of acetone were added to the water phase, and the extraction with isobutyl alcohol/benzene was repeated twice more. The water phase containing the radioactive glucose-6-p and ATP was mixed with 10 ml of water plus 1 ml of concentrated NH,OH and counted in the liquid scintillation counter. Rapid Mixing-This procedure was achieved by using a Durrum D-133 mixing device calibrated as previously described (26) or a Harvard Apparatus multispeed transmission device (22). The two instruments are equally suited to experimentation with reaction times of several milliseconds. However, the multispeed transmission device is limited to reaction times of approximately 10 to 300 ms. On the other hand, the Durrum mixer permits incubation times spanning between 17 ms and 10 s, through a combination of electronically controlled flow and aging procedures. In addition, the Durrum mixer permits the use of syringes of different volumes for rapid dilution experiments. Incubations were carried out at 25-28 C. with the aid of a temperature-controlled bath. RESULTS AND DISCUSSION Phosphorylation of SR Vesicles Exposed to EGTA Previous to Addition of P,-While enzyme phosphorylation with ATP is Ca +-dependent, enzyme phosphorylation with Pi requires removal of Ca from high affinity binding sites (8, 10, 12, 13). Accordingly, when P, is added to SR vesicles which are previously exposed to EGTA, rapid enzyme phosphorylation occurs (16). Maximal levels of phosphoenzyme are reached with a half-time of less than 20 ms (Fig. l.4). At saturating Pi, the phosphorylation reaction proceeds with first order kinetics, since P, concentrations yielding measurable amounts of phosphoenzyme are much higher than the enzyme concentration. On the other hand, initial velocities are dependent on the concentration of Pi and demonstrate saturation behavior, indicating the occurrence of a phosphate. enzyme complex previous to the phosphorylation reaction: E+P, 2 E.P, 2 E-P, k-1 k-p where velocity = &[E. Pi], and [E-P] = (kjk-2)[e. Pi]. Double reciprocal plots of final E-P levels as a function of P, concentrations (Fig. 1B) yield values of 2.0 nmol/mg for the equilibrium levels of E-P at saturating Pi concentrations and 1.1 mm for a K which is dependent on all four rate constants indicated in Equation 1. Loaded Vesicles-If SR vesicles are first loaded actively with calcium phosphate, centrifuged, and then resuspended in EGTA, subsequent addition of P, results in enzyme phospho- z 1.2 7 1.0 f 0.8! a c 0.6 5 6 = 0.4 k u: 0.2. 0. 4 I- K, FIG. 1. Phosphoenzyme formation following addition of Pi to Ca - loaded and nonloaded vesicles preincubated with EGTA. A, Nonloaded vesicles (0) or Ca +-loaded vesicles (0) were suspended in an EGTA-containing medium and, within a few seconds, the reaction was started by adding 12P, to a final concentration of 1 mm (0) or 0.8 mm (0). Other reactants and conditions are described under Experimental Procedures. B, double reciprocal plots of the final (30-s incubation) phosphoenzyme (E-P) levels as a function of P, concentration. Nonloaded (0) or Cap - loaded (0) vesicles were exposed to EGTA followed by reaction with various concentrations of P,. l/pi, (rr~m)-~

9466 A TPase Phosphorylation and A TP Synthesis rylation with a time constant (t1,2 = 320 ms) which is much slower (Fig. IA) than that observed with nonloaded vesicles, in agreement with the observations reported by Beil et al. (17). The P, concentration dependence is also different (Fig. lb), while the equilibrium level of E-P at saturating Pi is the same (18). These findings demonstrate that, following exposure to EGTA, P, addition to loaded or nonloaded vesicles results in phosphorylation of two different enzyme states, although most likely of the same site. Owing to relatively high solubility and dissociation of the calcium phosphate complex inside the vesicles, the different enzyme state observed in loaded SR may be attributed to Ca binding to low affinity sites exposed to the inner space of the vesicles: E + Ca,,, * E. Ca,,,. (2) In agreement with previous observations (16), some experiments with loaded vesicles reveal an initial fast component in the kinetics of phosphorylation. It is likely that this component is related to a fraction of the enzyme which is not bound to internal Ca +, when internal Ca is not maintained at saturating concentrations. Phosphorylation of SR Vesicles upon Simultaneous Addition of P, and EGTA-An interesting kinetic feature of the phosphorylation reaction is that if the SR vesicles are first exposed to low (-PM) Ca + concentrations, and then EGTA is added simultaneous with, rather than before Pi, a lag period is observed (Fig. 2). The occurrence of a lag period indicates that the enzyme containing calcium bound to high affinity sites undergoes a slow transition before becoming reactive to Pi (26): E. Caou, + E + Caour (3) E - *E -, where E is the reactive species which undergoes phosphorylation as in Equation 1. In similar experiments, Rauch et al. (16) observed a slow rate of phosphorylation when unloaded SR vesicles were preincubated with Ca )+ prior to the addition of EGTA and Pi. These authors, as well as DuPont and Leigh (27), have suggested that removal of Ca + from E.Cauut occurs through a multistep mechanism which, in itself, accounts for the slow transformation of E to *E. OJ +- 0 0.1 0.2 0.3 I +-r-+ I 0.4 0.6 1.0 FIG. 2. Phosphoenzyme formation following simultaneous addition of EGTA and Pi to vesicles preincuhated with Ca +. Nonloaded (0) or Ca -loaded (0) vesicles were suspended in a medium containing 50 pm CaClz, 20 mm Tris/maleate (ph 6.2), and 10 mm MgCI,. The reaction was started by addition of a solution containing EDTA and P,. After mixing, the final concentrations were 1.6 ITIM P,, 5 mm EGTA, 10 mm MgClr, 20 mm Tris/maleate (ph 6.2), 25 pm CaCll, and 0.6 mg of SR protein/ml. It is noteworthy that a lag period is not observed when the vesicles are exposed to EGTA before Pi addition (compare Figs. IA and 2), since the transition described in Equation 2 is already completed when Pi is added. Loaded Vesicles--In experiments in which EGTA and Pi are added simultaneously to vesicles which were first loaded actively with calcium phosphate, centrifuged, and resuspended in 50 pm CaC12, lag period and time dependence of phosphorylation with Pi are identical with those obtained with nonloaded vesicles (Fig. 2). Therefore, in these conditions, high intravesicular Ca2+ has no influence on the phosphorylation reaction, while displaying a marked effect when the vesicles are exposed to EGTA previous to Pi addition (compare Figs. IA and 2). In fact, it is expected that in the presence of Cap+ in the medium, nearly all the enzyme of both loaded and nonloaded vesicles is in the E ecaout form, owing to a much greater affinity constant of the E Cauut complex, as compared to that of the *E. Ca,, complex. Thereby, following simultaneous addition of EGTA and P,, E. Cauu, dissociates and E is transformed in *E which evidently reacts with P, before binding intravesicular Cap+: K ( 1,/,, E. Ca,,,, ti E + Ca,,, k2 E - *E k:j k:g *E + P, k~ *E.P, (4) I *E.P, k: *E-P 4 K ( */,/ *E-P + Ca,, e *E-P.Ca,,,, where *E indicates an enzyme species reactive to P,. It is then apparent that the kinetics of enzyme phosphorylation are dependent on the state of the enzyme wheu P, is added and not necessarily influenced by high intravesicular Ca +. Cleavage of the Phosphoenzyme Formed with P,-A most direct method to obtain a rate constant (Ke4 in Equation 4) for the hydrolytic cleavage of phosphoenzyme formed with P,, is dilution of the P, concentration after equilibrium E-P levels have been reached. In fact, if Pi is lowered to a concentration sustaining no or little phosphorylation, the E-P level decays to a low value with a time constant approximating kmq. It is found in these experiments (Fig. 3) that the time constant for decay of the E-P levels is 8 to 11 s-, a value quite similar to those obtained for hydrolytic cleavage of phosphoenzyme formed with ATP (26,28) and for the rate-limiting step of the Ca + pump in the forward direction (29). It is also found that identical time constants are obtained for the cleavage of E-P formed by adding EGTA and Pi either in sequence or simultaneously (Fig. 4), and independent of whether the vesicles are loaded with calcium (Fig. 3). This latter finding is quite interesting, considering that when the enzyme operates in the forward direction with ATP as a substrate, a slower turnover is observed in loaded as compared to unloaded vesicles, evidently due to significant reversal in the presence of the nucleotide. The observations reported above suggest that even though various kinetics of phosphoenzyme formation may be encountered in different experimental conditions, ultimately the same phosphoenzyme state undergoes hydrolytic cleavage. Such a state appears to be identical with that which is rate-limiting for the Ca + pump turnover in nonloaded vesicles. In addition to Pi dilution, experiments based on P, displace-

ATPase Phosphorylation and ATP Synthesis 9467 J, I I I I 0 0.2 0.4 0.6 10, FIG. 3. Phosphoenzyme decay induced by dilution of Pi. Phosphoenzyme formed from P, and nonloaded (0) or Ca -loaded (0) vesicles. Dephosphorylation was started by mixing 1 volume of a suspension containing 20 mm Tris/maleate (ph 6.2), 10 mm MgCb, 5 mm EGTA, 1.6 mm P,, and 1.2 mg of SR protein/ml with 9 volumes of 20 mm Tris/maleate (ph 6.2), 10 mm MgC12, and 5 mm EGTA. The reaction was quenched with acid at the times indicated. 1.6 2 a 5 h 1.2 e z 08 a E e P - 0 0.4 u; 0 I I I 0 1.0 2.0 70 FIG. 4. Decay of phosphoenzyme formed following simultaneous or sequential additions of EGTA and Ca +-loaded vesicles. Ca -loaded SR vesicles were suspended in EGTA-containing (0, n ) or Caz -containing (0, Cl) medium followed by hand-mixing addition of P, and EGTA to final concentrations of 1.6 and 2 mm, respectively. The phosphoenzyme suspension was placed in the rapid mixer syringe and the dephosphorylation was started by a lo-fold dilution of the. P, (0, n ), as in Fig. 3, or by mixing with an equal volume of a solution containing CaCb (0, 0) to give final concentrations of 1.5 mm CaCb, 1 mm EGTA, 20 mm Tris/maleate (ph 6.2), 10 mm MgCb, 1.6 mm P,, and 0.6 mg of SR protein/ml. Dephosphorylation was stopped with acid at the times indicated. ment by competition with added ATP (30) yield identical time constants for the hydrolytic cleavage of E-P. However, if decay of E-P is initiated by the addition of Ca2+ (500 pm) the resulting time constant is much slower (Fig. 4), in analogy with the slow phase observed by Rauch et al. (16) and de Meis and Tume (22). As the strategy of these latter experiments is to prevent further reaction of the dephosphorylated enzyme by calcium binding to the specific high affinity sites, the observed slow rate of net decay of E-P levels indicates that the reaction of the dephosphorylated enzyme with Pi is much faster than its transformation to a nonreactive species. Therefore, in Equation 4, kd is greater than k-2. It should be pointed out that addition of 20 IIIM EGTA and 30 IIIM CaCl2 induces a rapid decay of the phosphoenzyme levels (26), probably through complexation of phosphate by the high (IIIM) Cal+, or by nonspecific inhibition by the high EGTA.Ca levels (31). ATP Synthesis-A distinctive feature of the phosphoenzyme formed by reacting Pi with calcium-loaded vesicles is its ability to form ATP upon addition of ADP. On the other hand, the phosphoenzyme obtained in the absence of calcium loading cannot synthesize ATP. This inability, as well as the different kinetics of Pi incorporation, have raised the question of whether the phosphoenzymes formed with loaded or nonloaded vesicles are intermediates of the same reaction chain, or rather of parallel or branched pathways (23). In this regard, we have shown that phosphorylation of loaded or nonloaded vesicles can be obtained with identical kinetics (Fig. 2) simply by adding EGTA and Pi simultaneously, rather than in sequential order. Yet, even in these conditions, we found that ATP synthesis is only obtained with loaded vesicles. These findings indicate that addition of P, to loaded or nonloaded vesicles results in formation of the same phosphoenzyme which acquires the ability to synthesize ATP only in the presence of high intravesicular Ca. Bearing also on this point are experiments in which phosphorylation of loaded vesicles is obtained with different kinetics by varying the order of EGTA and P, addition (Fig. 5A). In both cases, ATP synthesis matches the transient kinetics of E-P formation (Fig. 5B) and then continues with steady state velocity when maximal levels of E-P are reached. These experiments show again that, independent of kinetic manipulations, a single phosphoenzyme is formed, which is readily utilized for ATP synthesis in the presence of high intravesicular Ca +. The experiments reported here do not distinguish whether high intravesicular Ca2+ or the presence of a transmembrane Ca + gradient is a requirement for ATP synthesis. It should be pointed out that single cycles of ATP synthesis were previously obtained by adding high Ca2+ and ADP to soluble phosphoenzyme (15, 22). Therefore, it would appear that while high intravesicular Ca + is a primary requirement for ATP synthesis, low Ca + in the outside medium is necessary to permit further enzyme phosphorylation and steady state ATP synthesis. CONCl.USIONS Our findings indicate that various kinetic features of enzyme phosphorylation with Pi are related to different states in which 1.61 A 1.61 B FIG. 5. Early phase of phosphoenzyme formation and ATP synthesis in Ca +-loaded vesicles pre-exposed to EGTA or Ca +. Ca -loaded SR vesicles were suspended in EGTA-containing (0) or Ca -containing (0) medium and the reaction was started by simultaneous addition of EGTA, P,, and ADP. Following acidquenching the phosphoenzyme levels (A) and ATP synthesized (B) were measured. Other conditions were described under Experimental Procedures.

I. 9468 ATPase Phosphorylation and ATP Synthesis the enzyme may reside when Pi is added. This can be iiiustrated by considering the following reaction scheme: EGTA. CA,,, EGTA ATP E+-Ed&G-+ ATP. E. CaUUt c--) t 1 8 7 L 3 ADP. E-P. Caout *E.P, <4_\ *E-P& *E-z.Cai, lb!& *E.Ca,,, 4+P,.E.Ca,,, 2-J SCHEME 1 It is apparent from this scheme that if Pi is added to vesicles previously exposed to EGTA, the enzyme is already deprived of Caa+ and the reactive form *E is phosphorylated directly through Steps 3 and 4, or at a slower rate through Steps 10 and 11, if the vesicles are loaded with calcium (Fig. 1). On the other hand, in the presence of sufficient Cd:.:,, nearly aii the enzyme of nonloaded or loaded vesicles is in the E - Caout form. Therefore, upon simultaneous addition of EGTA and P,, Caa* dissociation and E conversion to *E (Steps 1 and 2) must proceed Steps 3 and 4, thereby influencing the kinetics of phosphorylation (Fig. 2). A comparison of Figs. IA and 2 suggests that upon conversion of E to *E, the phosphorylation reaction precedes binding of Cafz. It is noteworthy that hydrolytic cleavage of phosphoenzyme formed with Pi (Fig. 3) is consistent with the turnover of Caa+- ATPase in the presence of low intravesicular Ca. Furthermore, the slow decay of phosphoenzyme observed after addition of Caa+ to the medium (Fig. 4) indicates that reversal of *E to E (Step 2) is slower than the phosphorylation reaction (Steps 3 and 4). Finally, in the presence of high intravesicular Ca + and independent of the kinetics of phosphorylation (Fig. 5), *E-P. Cai, can be utilized through Steps 6 and 7 for synthesis of ATP. 1. 2. 3. 4. 5. 6. 7. REFERENCES Yamamoto, T., and Tonomura, Y. (1967) J. Biochem. (Tokyo) 62, 558-575 Makinose, M. (1969) Eur. J. Biochem. 10, 74-82 Martonosi, A. (1969) J. Biol. Chem. 244,613-620 Inesi, G., Maring, E., Murphy, A. J., and McFarland, B. H. (1970) Arch. Biochem. Biophys. 138,285-294 Inesi, G., Kunmack, M., and Verjovski-Ahneida, S. (1978) Ann. N. Y. Acad. Sci. 307,224-227 Ikemoto, N. (1976) J. Biol. Chem. 251, 7275-7277 Barlogie, B., Hasselbach, W., and Makinose, M. (1971) FEBS Lett. 12,267-268 8. Makinose, M., and Hasselbach, W. (1971) FEBS Lett. 12, 271-272 9. Makinose, M. (1972) FEBS Lett. 25, 113-115 10. Yamada, S., Sumida, M., and Tonomura. Y. (1972) J. Biochem. (Tokyo) 72, 1537-1548 11. Yamada. S.. and Tonomura. 1091-i986 Y. (1973) J. Biochem. (Z okvo). 74., 12. Kanazawa, T., and Boyer, P. D. (1973) J. Biol. Chem. 248,3163-3172 13. 14. 15. Masuda, H., and de Meis, L. (1973) Biochemistry 12,4581-4585 Kanazawa, T. (1975) J. Biol. Chem. 250, 113-119 Knowles, A. F., and Racker, E. (1975) J. Biol. Chem. 250, 1949-1951 16. Rauch, B., Chak, D. V., and Haaaelbach, W. (1972) 2. Naturforsch. 32c, 828-834 17. BeiI, F. V., Chak, D. C., and Hasselbach, W. (1977) Eur. J. Biochem. 81, 151-164 18. 19. de Meis. L. (1976) J. Biol. Chem. 251. 2055-2062 de Meis; L., and darvalho, M. G. C. (1974) Biochemistry 13,5032-5038 20. de Meis, L., and Sorenson, M. M. (1975) Biochemistry 14, 2739-2744 21. Carvalho, M. G. C., Souza, D. 0. G., and de Meis, L. (1976) J. Biol. Chem. 251,3629-3636 22. 23. 24. de Meis, L., and Tume, R. K. (1977) Biochemistry 16,4455-4463 Hasselbach, W. (1978) Biochim. Biophys. Acta 463,219-302 de Meis, L., and Hasselbach, W. (1971) J. Biol. Chem. 246,4759-4763 25. Lowry, 0. H., Rosebrough, N. J., Fan, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 26. Verjovski-Almeida, S., Kurzmack, M., and Inesi, G. (1978) Biochemistry 17, 5006-5013 27. 28. 29. 30. DuPont, Y., and Leigh, J. B. (1978) Nature 273.393-398 Froehlich, J. P., and Taylor, E. W. (1975) J. Biol. Chem. 250, 2013-2021 Inesi, G., and Scarpa, A. (1972) Biochemistry 11.356-359 Vievra. A.. Scofano. H. M.. Guimaraes-Motta. H.. Tume. R. K.. and de Meis, L. (1979) Blochim. Biophys. Acta, in press 31. Verjovski-Almeida, S., and De Meis, L. (1977) Biochemistry 16, 329-334