Eur. J. Biochem. 193, 535-539 (1990) 0FEBS 1990
Blockage of a pump-related calcium-efflux pathway in light sarcoplasmic reticulum vesicles by Mops Sibylle STEPHAN, Andrea MIGALA and Wilhelm HASSELBACH Max-Planck-Institut fur Medizinische Forschung, Abteilung fur Physiologie, Heidelberg, Federal Republic of Germany (Received July 11, 1990) - EJB 900832
Mops, used as a proton buffer, specifically enhances the accumulation of calcium or strontium by light sarcoplasmic reticulum vesicles driven by ATP or dinitrophenylphosphate as energy-yielding substrates when calcium-precipitating agents are absent. The enhancement of ion uptake by Mops is much greater for strontium than for calcium and is further increased when potassium is replaced by sodium as the dominant monovalent cation. Mops affects neither the activity of the calcium- or strontium-activated transport enzyme nor the active accumulation of calcium in the presence of oxalate, i.e. when the pump runs unidirectionally forward. Passive calcium and strontium efflux rates of approximately 40- 50 nmol . mg-' . min-l are considerably reduced when histidine/glycerophosphate or Tris/maleate are exchanged for Mops. The observed passive efflux rates and their modulation by Mops are too small, in relation to the rate of ion influx, to account for either the relatively small calcium and strontium load in the absence of precipitating agents or for its modulation by Mops. The results imply that the pump itself mediates ion efflux dependent on pump activity and the different degree of saturation of lumenal ion-binding sites by calcium and strontium, as well as their susceptibility to Mops. It is well established that the sarcoplasmic reticulum calcium-transporting ATPase is inhibited by calcium concentrations in the millimolar range [l, 21. The dependence of the inhibitory effect of calcium on the occupancy of lumenal binding sites of native sarcoplasmic reticulum vesicles was recognized quite early [3]. The activation of enzymatic and transport activity of native sarcoplasmic reticulum vesicles in the presence of oxalate was explained by the formation of calcium oxalate precipitates inside the vesicles counteracting the elevation of the internal calcium level. In the absence of precipitating anions such as oxalate or phosphate, the rapid rise in internal calcium truncates enzymatic and transport activity, limiting the calcium-uptake capacity to approximately 100- 150 nmol/mg. An internal free-calcium concentration of 20mM can be estimated from this amount [4]. This high calcium concentration, which counteracts calcium accumulation, is an essential prerequisite for the calciumefflux-driven ATP synthesis in the reverse mode of the pump [5].When the internal calcium concentration has reached its maximal level, a small but measurable calcium-dependent ATPase activity persists which was correlated to a residual calcium influx by Waas and Hasselbach [6]. This persisting influx was assumed to be balanced by an efflux of calcium, mediated by the pump itself and not by unspecific membrane leaks. Ion exit was supposed to be coupled to the transition from the low-affinity (E,) to the high-affinity (El) state of the enzyme. This reaction step was discussed by Johnson et al. [7] as an alternative pathway of the reaction cycle to shift the pump stoichiometry (calcium/ATP) from 2: 1 to 1 :1. Correspondence to W. Hasselbach, Max-Planck-Institut fur Medizinische Forschung, JahnstraBe 29, W-6900 Heidelberg, Federal Republic of Germany Abbreviation. DnpP, dinitrophenylphosphate. Enzyme. Calcium-transporting ATPase (EC 3.6.1.38)
It is shown in this report that the enzyme is not inhibited by the accumulated ions (trans-inhibition) if the physiological transport substrate, calcium, is exchanged for its congener strontium, although high internal strontium concentrations are reached [8, 91. The pathway by which ions escape from the vesicle, after strontium uptake has reached a steady state, is specifically blocked by Mops buffer in combination with sodium ions. Calcium-release channels, present in heavy sarcoplasmic reticulum vesicles, do not contribute to this effect. These are only a minor contaminant in the light sarcoplasmic reticulum vesicle preparation and are inactivated by high magnesium concentrations (10 mM) used in these experiments [lo, 111. Therefore our findings support the true existence of a highly specific pathway for ion exit in light sarcoplasmic reticulum vesicles, which is most probably a part of the reaction cycle. MATERIALS AND METHODS Enzyme preparation
Light sarcoplasmic reticulum vesicles were prepared from rabbit skeletal muscle, as described by de Meis and Hasselbach [QI. Enzyme assays
Dinitrophenylphosphatase (DnpPase) activity of calciumpermeable sarcoplasmic reticulum vesicles was measured in media containing 0.1 M NaCl or KCl, 20 - 50 mM histidine/ glycerophosphate or 30-50 mM Tris/Mops, pH 7.0, 0.1 M sucrose, 2 pM calcium ionophore A23187,lO mM MgC12 and 1 mM DnpP. For measuring the activity of native vesicles, no calcium ionophore was added to the media. The concentrations of calcium and strontium are specified in the legends
536 of figures and tables, together with the concentrations of sodium or potassium ions and the respective buffers. The concentration of the vesicles in the assay media were chosen between 0.01 mg/ml and 0.05 mg/ml. depending on enzyme activity under the respective conditions. t = 20°C. For measuring the dependence of DnpP hydrolysis on calcium and strontium, the reaction was terminated with sodium dodecyl sulfate, l % final concentration, at appropriate times. Activity was calculated by using an E~~~ of 6600 M-' . cm- ', determined with 2,4-dinitrophenol under the same conditions [I 31. Calcium and strontium uptake were measured in media of the following composition: 1 mM DnpP, 10 mM MgCI2, 0.1 M KCI or NaC1. 40 mM histidine/glycerophosphate, 50 mM K '/Mops, or 50 mM Trisjmaleate, 20 pM 90Sr2+or 45Ca2t(300 Bqinmol) and 0.05 mgvesicle/ml. The uptake was monitored for 20 min. At chosen time intervals, aliquots of 5 ml were removed and filtered through Schleicher & Schiill filters BA85, 0.45 pin. ATP-supported uptake was measured in media containing 2 mM ATP instead of DnpP. For measuring calcium or strontium release, the vesicles were at first loaded under the same conditions as those described for calcium and strontium uptake, except that the DnpP concentration was reduced to 0.6 mM. This low substrate concentration is completely used up during loading. Thus the subsequent release proceeds purely passively. After 15 min, one aliquot (40 ml) was added to 3 vol. release medium. This contained 0.1 M NaCl or KCl, 10 mM MgCI2, 50 mM histidine/glycerophosphate or 50 mM TrislMops. Aliquots were taken and filtered through Schleicher & Schiill BA85,0.45 pm, at appropriate time intervals. For both uptake and release of calci um and strontium, appropriate proteinfree blanks were tested by filtering protein-free solutions. The calcium-transport ratio was determined by monitoring the burst of dinitrophenol liberated following the addition of 3 - 10 nmol/ml calcium to the assay medium containing 0.1 M KCI, 0.1 M sucrose, 10 mM MgC12, 5 mM oxalate, 25 mM Tris/Mops or Tris/maleate, pH 7, and 0.3 mM DnpP. Reagents
Lutidine DnpP was synthesized by Mr. H. Gaukler according to [14] in our laboratories. A23187 was purchased from calbiochem GmbH, Frankfurt, FRG; other reagents were bought from Serva, Heidelberg, FRG, and Sigma, Deisenhofen, FRG.
RESULTS The dependence on calcium and strontium concentrations of the activity of the calcium transport enzyme was first analysed with A23 187-treated light sarcoplasmic reticulum vesicles using DnpP as substrate. The enzyme is activated by calcium in the same concentration range, 0.1 - 1.O pM, as that reported for calcium-dependent ATP hydrolysis 1131. Strontium activation was measured in the presence of 0.5 mM EGTA to suppress activation by contaminating calcium [8]. This is possible because the affinity of EGTA for strontium is lower by a factor of 1000 than that for calcium. The dissociation constant of strontium-EGTA was determined with an ion-sensitive electrode under our experimental conditions. The obtained value of 0.1 mM agrees quite well with the dissociation constant reported by Schwarzenbach et al. [I 51. On account of the great difference between the dissociation
0.03
a 1.0 10 0
02 Strontium (mM)
Strontium * 0 - ~ 0 rCclcium I O - ' ( ~ V )
Fig. 1. Strontium activation of' DnpP hydro1,vsis by sarcoplusmic reticulum vesicles. The assays contained 1 niM DnpP, 10 m M MgCIZ, 20 mM histidine/glycerophosphate, 0.1 M NaCI, 0.5 m M EGTA, 0.1 mgjmlvesicularprotein, 2 pM A23187; t = 20°C. (A) Dependence of strontium-activated DnpP hydrolysis on free strontium. (B) Hill plot of strontium-activated DnpP hydrolysis (A)and comparison of calcium-activated hydrolysis (M). A and A,,, are the actual and maximal enzyme activities, respectively 600 1
0.600 1.000
10.000 Calcium or Strontium (mM)
Fig. 2. Inhibition by strontium or calcium of DnpP hydrolysis of'sarcoplasmic reticulum vesicles. The assays contained 1 mM DnpP, 10 mM MgCIZ, 20 mM histidine/glycerophosphate ( A , 0) or 50 mM Tris/ Mops (A , O ) ,0.1 M NaC1,O.l mg/ml vesicular protein, 2 pM A23187 and the concentrations of calcium and strontium shown. Strontiumactivated DnpP hydrolysis was measured in the presence of 0.5 mM EGTA (A,A). Calcium-dependent DnpP hydrolysis (0.0).t = 20°C constant of calcium- and strontium-EGTA, EGTA complexes only very little of the added strontium while calcium contamination of the system is reduced below activating levels. As shown in Fig. 1 A and B, strontium activation occurs in the submillimolar range (K, = 0.3 mM) having a relatively flat slope resulting in a Hill coefficient of 1.2, which is markedly smaller than that obtained for calcium activation. Inhibition of the calcium-transport enzyme by calcium ion concentrations exceeding 0.1 mM is a characteristic feature of the enzyme. Fig. 2 shows that enzyme activity is suppressed by 50%)at a concentration of 1 mM and that its decline occurs within one order of magnitude with respect to concentration [16, 171. In contrast to calcium, 20 mM strontium does not significantly interfere with DnpP hydrolysis. Even in the presence of 30 mM strontium (not shown), enzyme activity is only partially suppressed. Yet the effect of high concentrations of strontium is difficult to judge because of almost unpredictable interference with the ion composition of the system. Fig. 2 further illustrates that the observed activity pattern does not depend on the nature of the pH buffer used in these experiments.
537 Table 1. Effect of different proton buffers on the DnpP hydrolysis of permeable and native surcoplasmic reticulum vesicles activated by calcium or strontium in Nu+-containing media. The assays contained 1 mM DnpP, 10 mM MgC12, 0.1 M NaCl, 0.2 mM CaCI2 or 3.0 mM SrC1, with 0.5 mM EGTA, the listed buffers and 0.02 mg/ml permeable light vesicles; t = 20°C. The rates are given as means f SEM; n = 6 Proton buffer
DnpPase activation by strontium of permeable vesicles
calcium of native vesicles
permeable vesicles
native vesicles
106.3 _+ 7.48 (n = 6)
550.5 _+ 8.7 (n = 8)
52.8 & 3.75 ( n = 6) 75.5 & 4.8 ( n = 7)
nmol . mg-' . min-' Tris/Mops (50 mM)
414.4 f 8.11 (n = 8)
Histidine/glycerophosphate (20 mM)
475.8
20.3 (n = 7)
283.5 k 16.6 ( n
=
7)
565.0 5 24.0 ( n
Tris/maleate (20 mM)
435.9 & 13.2 (n = 3)
264.5 k 16.04 (n
=
3)
-
The observation that high concentrations of calcium do, while those of strontium do not, produce measurable inhibition of DnpP hydrolysis of permeable vesicles, is in line with the enzymatic behavior of closed native vesicles. At calcium concentrations whch optimally activate the DnpP hydrolysis of calcium-permeable vesicles, the hydrolysis of closed vesicles reaches less than 20% of maximal activity, irrespective of the buffer composition of the media. Strontium-activated DnpP hydrolysis by closed vesicles behaves quite differently with respect to extent and buffer sensitivity of trans-inhibition. In the presence of histidine/glycerophosphate or of Tris/maleate the activity of closed vesicles is only 40% lower than that of calcium-permeable vesicles. In contrast, when the system is buffered with Tris/Mops or sodium/Mops, the activity of the closed vesicles drops to 25% of that of permeable control preparations (Table 1). The data given in Table 1 show that buffer composition affects the extent of trans-inhibition by calcium much less than that produced by strontium. This finding suggests that strontium storage produces strong inhibition only in the presence of Tris/Mops, while calcium storage reaches inhibitory concentrations with all applied buffers. The marked difference in the effects of the respective buffers on enzyme activity of the closed vesicles indicates that the buffers might interfere with the ion-storing capacity of the vesicle preparations. Uptake experiments performed under corresponding conditions furnished the expected results, and in addition revealed that the enhancement of ion uptake by Mops markedly depends on whether sodium or potassium ions are present as monovalent cations. In the presence of sodium, strontium uptake energized by DnpP as well as by ATP is strongly enhanced in the presence of Mops, as compared to histidine or Tris/maleate. If potassium is the prevalent monovalent ion, uptake activation produced by Mops is less pronounced (Table 2). In contrast, calcium uptake is less affected by medium composition with respect to buffers and monovalent cations. Calcium uptake in Mops-buffered media is twofold higher than in histidine-buffered or Tris/maleate-buffered media, irrespective of the presence of sodium or potassium. The uptake transients observed for ATP-supported uptake do not interfere with our reasoning (not shown). Similar transients were reported by Sorenson and de Meis [18] in Tris/maleate-buffered media at pH 6.0. The large effects which proton buffers and ions exert on strontium and calcium-storing capacity cannot be related to alterations in the activity of the forward-running ion pump, as revealed by small variations in enzyme activity
=
11)
-
Table 2. Dependence of ion storage on energy supply, monovalent cations and proton buffers. Ion storage was determined after an uptake period of 15 min in the assays given in Materials and Methods. The assays containing 1 mM DnpP or 2 mM ATP, 10 mM MgCI,, the listed buffers, 0.1 M NaCl or 0.1 M KCl, 20 pM 45Ca or "Sr and 0.05 mg/ml native light vesicles; t = 20°C Ion stored Proton buffer
Energy supplied by DnpP with
Na+
ATP with K'
Na'
K+
nmol/mg Strontium Histidine/ glycerophosphate (20 mM) Mops (50 mM) Calcium
10 60-
70
15 5
Histidine/ glycerophosphate 60- 70 60 (20 mM) Mops(50mM) 100-110 120
30 50-90
10 20-35
60
50
120
100
of leaky vesicles under corresponding conditions (Table 1). Therefore, it appears likely that buffers and ions might inhibit calcium efflux. To measure calcium efflux, the vesicles were at first actively loaded for 10 - 20 min with strontium or calcium using DnpP as the energy donor. Subsequently, release was initiated by diluting 1 vol. uptake medium with 3 vol. of the corresponding release media containing sodium as the main cation. The results illustrated in Fig. 3A and B show that calcium and strontium efflux occur much faster in histidine/ glycerol than in Mops-buffered media. The same is true, to a lesser extent, if sodium as the main cation in the release medium is exchanged for potassium (Table 3).
DISCUSSION The results presented in this study show that the pathway for calcium and strontium efflux through the membranes of light sarcoplasmic reticulum vesicles is specifically blocked when Mops is used as the proton buffer instead of other buffers, e. g. histidine/glycerophosphate or Tris/maleate. Calcium-releasing channels, present in heavy sarcoplasmic reticulum vesicles, are absent in the light preparations used and are blocked by the presence of 10 mM magnesium [lo].
538 A
B
storage are enhanced by the buffer. Like passive calcium or strontium efflux, the enhancement of ion uptake by Mops as 0compared to histidine/glycerophosphate depends on whether I the uptake medium contains mainly sodium or potassium E ions. In general, calcium uptake and release are considerably 0 0 80 E less affected by Mops than the corresponding strontium move60.v ments. Sodium in the medium augments the effect of Mops E P A on storage and release of both ions. 40.The observed rates of passive efflux in the absence as well 5+. ._ E as in the presence of Mops (Fig. 3, Table 3) are at variance 2 20.with the high rates of calcium or strontium-supported DnpP A .-A -' hydrolysis persisting after ion uptake (Table 1) has reached 10 15 20 25 30 steady state. Since this activity has been shown to be directly 10 15 20 25 30 Time (mi.) related to calcium or strontium turnover 16, 81, we have to T'me ( m i l ) Fig. 3. Calcium or strontium release modified by medium buffers. The assume that during active accumulation in the absence of conditions for measuring the release are given in Materials and precipitating ions that an ion efflux must occur which is much Methods. The media in (A) and (B) contained 0.1 M NaCl and (A) faster than the observed rates of passive release. This rapid 20 mM histidine/glycerophosphate or ( 0 )50 mM Tris/Mops, respec- ion efflux is most likely mediated by the active pump itself. tively. (A) Calcium release; (B) strontium release Such an ion-exit pathway could prevent overloading of the sarcoplasmic reticulum if the amount of ions offered for storage exceeds the physiological storage capacity which Tdbk 3. EJfect of' different proton buffers on the rate of calcium or might be limited by the amount of binding proteins 1231 and strontium releu.c.e.from actively loaded sarcoplasmic reticulum vesicles by the level of inorganic phosphate [16, 241. The existence of in sodium- or pota,r.riiim-contailzin~media. The composition of the such a pump-related efflux in sarcoplasmic reticulum memassays for calcium or strontium loading is described in Materials and Methods. The release media contained 0.1 M NaCl or 0.1 M KCI, branes has been proposed by Waas and Hasselbach [6] and 10 mM MgCI2 and the listed buffers. Release was initiated by diluting discussed in relation to pump efficiency by Johnson et al. [7]. 1 vol. uptake medium with 3 vol. release medium It remains to be established if the slow passive efflux is also mediated by the pump but in a non-energized mode. Ion stored Proton buffer Initial rate of ion release An efflux mediated by the active pump would lead to a fast turnover of the energy donors ATP or DnpP at the cessation of K' Na net calcium uptake, and thus to continuous energy dissipation. Yet, in the case of calcium storage, this situation does not nmol. mg-' . min-' occur because the transport enzyme is inhibited when the calcium concentration at the lumenal surface reaches 3 Strontium Histidine glyccrophosphate 5 mM, which is rapidly reached inside native vesicles in the (20 mM) 40 31 absence of precipitating ions [17]. Mops (50 mM) 15 30 The reasoning above can also be applied to account for the behavior of the transport system during strontium acCalcium Hist idinc glycerophosphdte cumulation and its strong modulation by Mops. In contrast (20 m M ) 20 45 to calcium, strontium does not inhibit the enzymatic activity Mops (50 mM) 10 25 of the pump enzyme, even in the presence of 20- 30 mM, which is at least 10 times higher then the inhibitory concentration of calcium. Hence strontium, in contrast to calcium accumulation, is not truncated by pump inhibition. Evidently Therefore they are not part of the calcium-efflux pathway the lumenal binding sites of the enzyme related to pump inhiaffected by Mops. Mops affects neither the calcium- nor the bition have a much lower affinity for strontium than for strontium-activated hydrolysis of ATP or DnpP. Further- calcium. Since in the absence of Mops the proceeding active more, it has repeatedly been shown that Mops does not inter- strontium uptake only results in a small steady-state strontium fere with ATP-supported calcium uptake in the presence of the load (compare Table 2), we have to assume that strontium, calcium precipitating agent, oxalate. ATP-supported-calcium- although its concentration remains quite low, can rapidly be uptake measurements in the presence of Mops [19,20] yielded carried outwards, as already shown by Mermier and rates which fully agree with those observed in the presence Hasselbach [8]. Mops evidently interferes with this reaction. of other buffers [I, 211. Furthermore, calcium uptake in the The marked inhibition of DnpP hydrolysis by closed native presence of oxalate supported by DnpP, which was first de- vesicles can only be produced by a significant increase of scribed by Rossi et al. [22], furnished the same transport ratio internal strontium exceeding 30 mM. Strontium concen(calcium taken up)/DnpP hydrolysed) of 1.4 in media buffered trations inside the vesicles, in the order of 0.3 M, have been with either Mops or Trislmaleate. Thus, we can presume that reported for ATP-dependent strontium storage by Mermier the coupling between substrate hydrolysis and ion translo- and Hasselbach [8]. These concentrations could be reached cation is not differently affected by the buffers. under our experimental conditions selected for measuring enIn the absence of oxalate, where the internal ion concen- zyme activity if only 1% of the added strontium is stored by trations rapidly reach quite high values, the interference of the vesicles. Mops with ion storage becomes manifest, whereby the storage As already mentioned, the effect of Mops on calcium acof strontium is affected to a much larger extent than that of cumulation, and calcium activated steady-state DnpP hycalcium. The retardation of passive calcium efflux produced drolysis by native vesicles, is much smaller than that on the by Mops is in line with the finding that calcium and strontium corresponding reaction with strontium. This can most likely 1007
4
20
\
'\'
y1
c
v)
+
539 be related to the low rate of calcium turnover resulting from severe trans-inhibition. A further compensatory reduction of the rate of calcium efflux would only lead to a small increase of the lumenal free-calcium concentration, and as a consequence to a small increment of bound calcium. As for the role of monovalent cations in the effect of Mops, specific differences between calcium and strontium storage have been observed. The enhancement of calcium storage by Mops is the same in the presence of sodium and potassium (compare Table 2), and does not depend on the activity-yielding substrate. In contrast, Mops distinctly enhances strontium uptake only if sodium is the main monovalent cation. This indicates that the efflux of calcium and strontium through the active pump depends in a rather complex manner on the interaction of the transport enzyme with various ionic components, buffers, monovalent and divalent ions. We are indebted to Mr. A. Pfandke for his help in preparing the sarcoplasmic reticulum vesicles.
REFERENCES 1. Makinose, M. & Hasselbach, W. (1965) Biochem. Z. 343, 360382. 2. Weber, A,, Herz, R. & Reiss, I. (1966) Biochem. Z. 345, 329369. 3. Hasselbach, W. (1966) Ann. N. Y . Acad. Sci. 137, 1041- 1048. 4. Hasselbach, W. & Oetliker, H. (1983) Annu. Rev. Physiol. 45, 125- 139. 5. Makinose, M. & Hasselbach, W. (1971) FEBS Lett. 12, 271272.
6. Waas, W. & Hasselbach, W. (1981) Enr. J . Biochem. 116, 601 608. 7. Johnson, E. A., Tanford, Ch. & Reynolds, J. A. (1985) Proc. Nut1 Acad. Sdi. U S A 82, 5352 - 5356. 8. Mermier, P. & Hasselbach, W. (1976) Eur. J. Biochem. 69, 7986. 9. Guimares-Motta, H., Sande-Lemos, M. P. & de Meis L. (1984) J . Biol. Chem. 259, 8699-8705. 10. Meissner, G. (1984) J . Bid. Chem. 259, 2365 - 2374. 11. Su, J. & Hasselbach, W. (1984) Pfliigers Arch. 400, 14-21. 12. de Meis, L. & Hasselbach, W. (1971) J . Biol. Chem. 246, 47594163. 13. Hasselbach, W. (1988) Z . Nuturforsch. 43c, 929-937. 14. Ramirez, F. & Maracek, J. F. (1978) Synthesis 5, 601 -603. 15. Schwarzenbach, G. (1960) Die komplexometrische Titration, Ferdinand Enke Verlag, Stuttgart. 16. Inesi, G. & de Meis, L. (1989) J . Biol. Chem. 264, 5929-5936. 17. Hasselbach. W. (1976) Molecular bnsis of motility (Heilmeyer, L. M. G. et al., eds) pp. 81 -92, Springer V. Berlin, Heidelberg. 18. Sorenson, M. M. & de Meis, L. (1917) Biochim. Biophys. Actu 465,210-223. 19. de Meis, L. & Sorenson, M. M. (1989) Biochim. Biophys. Actu 984, 313 - 378. 20. Gafni, A. & Boyer, P. D. (1985) Proc. Natl Acad. Sci. USA 82, 89-101. 21. Hasselbach, W. & Migala, A. (1985) Z. Naturforsch. 40c, 571 575. 22. Rossi, B., Leone, F., Gacho, Ch. & Lazdunski, J. M. (1979) J . Biol. Chem. 254, 2302 - 2307. 23. MacLennan, D. H., Ostwald, T. J. & Stewart, P. S. (1973) Ann. N . Y. Acad. Sci. 227,527- 536. 24. Fassold, E. & Hasselbach, W. (1986) Eur. J . Biochem. 154, 714.
Blockage of a pump-related calcium-efflux pathway in light sarcoplasmic reticulum vesicles by Mops Sibylle STEPHAN, Andrea MIGALA and Wilhelm HASSELBACH Max-Planck-Institut fur Medizinische Forschung, Abteilung fur Physiologie, Heidelberg, Federal Republic of Germany (Received July 11, 1990) - EJB 900832
Mops, used as a proton buffer, specifically enhances the accumulation of calcium or strontium by light sarcoplasmic reticulum vesicles driven by ATP or dinitrophenylphosphate as energy-yielding substrates when calcium-precipitating agents are absent. The enhancement of ion uptake by Mops is much greater for strontium than for calcium and is further increased when potassium is replaced by sodium as the dominant monovalent cation. Mops affects neither the activity of the calcium- or strontium-activated transport enzyme nor the active accumulation of calcium in the presence of oxalate, i.e. when the pump runs unidirectionally forward. Passive calcium and strontium efflux rates of approximately 40- 50 nmol . mg-' . min-l are considerably reduced when histidine/glycerophosphate or Tris/maleate are exchanged for Mops. The observed passive efflux rates and their modulation by Mops are too small, in relation to the rate of ion influx, to account for either the relatively small calcium and strontium load in the absence of precipitating agents or for its modulation by Mops. The results imply that the pump itself mediates ion efflux dependent on pump activity and the different degree of saturation of lumenal ion-binding sites by calcium and strontium, as well as their susceptibility to Mops. It is well established that the sarcoplasmic reticulum calcium-transporting ATPase is inhibited by calcium concentrations in the millimolar range [l, 21. The dependence of the inhibitory effect of calcium on the occupancy of lumenal binding sites of native sarcoplasmic reticulum vesicles was recognized quite early [3]. The activation of enzymatic and transport activity of native sarcoplasmic reticulum vesicles in the presence of oxalate was explained by the formation of calcium oxalate precipitates inside the vesicles counteracting the elevation of the internal calcium level. In the absence of precipitating anions such as oxalate or phosphate, the rapid rise in internal calcium truncates enzymatic and transport activity, limiting the calcium-uptake capacity to approximately 100- 150 nmol/mg. An internal free-calcium concentration of 20mM can be estimated from this amount [4]. This high calcium concentration, which counteracts calcium accumulation, is an essential prerequisite for the calciumefflux-driven ATP synthesis in the reverse mode of the pump [5].When the internal calcium concentration has reached its maximal level, a small but measurable calcium-dependent ATPase activity persists which was correlated to a residual calcium influx by Waas and Hasselbach [6]. This persisting influx was assumed to be balanced by an efflux of calcium, mediated by the pump itself and not by unspecific membrane leaks. Ion exit was supposed to be coupled to the transition from the low-affinity (E,) to the high-affinity (El) state of the enzyme. This reaction step was discussed by Johnson et al. [7] as an alternative pathway of the reaction cycle to shift the pump stoichiometry (calcium/ATP) from 2: 1 to 1 :1. Correspondence to W. Hasselbach, Max-Planck-Institut fur Medizinische Forschung, JahnstraBe 29, W-6900 Heidelberg, Federal Republic of Germany Abbreviation. DnpP, dinitrophenylphosphate. Enzyme. Calcium-transporting ATPase (EC 3.6.1.38)
It is shown in this report that the enzyme is not inhibited by the accumulated ions (trans-inhibition) if the physiological transport substrate, calcium, is exchanged for its congener strontium, although high internal strontium concentrations are reached [8, 91. The pathway by which ions escape from the vesicle, after strontium uptake has reached a steady state, is specifically blocked by Mops buffer in combination with sodium ions. Calcium-release channels, present in heavy sarcoplasmic reticulum vesicles, do not contribute to this effect. These are only a minor contaminant in the light sarcoplasmic reticulum vesicle preparation and are inactivated by high magnesium concentrations (10 mM) used in these experiments [lo, 111. Therefore our findings support the true existence of a highly specific pathway for ion exit in light sarcoplasmic reticulum vesicles, which is most probably a part of the reaction cycle. MATERIALS AND METHODS Enzyme preparation
Light sarcoplasmic reticulum vesicles were prepared from rabbit skeletal muscle, as described by de Meis and Hasselbach [QI. Enzyme assays
Dinitrophenylphosphatase (DnpPase) activity of calciumpermeable sarcoplasmic reticulum vesicles was measured in media containing 0.1 M NaCl or KCl, 20 - 50 mM histidine/ glycerophosphate or 30-50 mM Tris/Mops, pH 7.0, 0.1 M sucrose, 2 pM calcium ionophore A23187,lO mM MgC12 and 1 mM DnpP. For measuring the activity of native vesicles, no calcium ionophore was added to the media. The concentrations of calcium and strontium are specified in the legends
536 of figures and tables, together with the concentrations of sodium or potassium ions and the respective buffers. The concentration of the vesicles in the assay media were chosen between 0.01 mg/ml and 0.05 mg/ml. depending on enzyme activity under the respective conditions. t = 20°C. For measuring the dependence of DnpP hydrolysis on calcium and strontium, the reaction was terminated with sodium dodecyl sulfate, l % final concentration, at appropriate times. Activity was calculated by using an E~~~ of 6600 M-' . cm- ', determined with 2,4-dinitrophenol under the same conditions [I 31. Calcium and strontium uptake were measured in media of the following composition: 1 mM DnpP, 10 mM MgCI2, 0.1 M KCI or NaC1. 40 mM histidine/glycerophosphate, 50 mM K '/Mops, or 50 mM Trisjmaleate, 20 pM 90Sr2+or 45Ca2t(300 Bqinmol) and 0.05 mgvesicle/ml. The uptake was monitored for 20 min. At chosen time intervals, aliquots of 5 ml were removed and filtered through Schleicher & Schiill filters BA85, 0.45 pin. ATP-supported uptake was measured in media containing 2 mM ATP instead of DnpP. For measuring calcium or strontium release, the vesicles were at first loaded under the same conditions as those described for calcium and strontium uptake, except that the DnpP concentration was reduced to 0.6 mM. This low substrate concentration is completely used up during loading. Thus the subsequent release proceeds purely passively. After 15 min, one aliquot (40 ml) was added to 3 vol. release medium. This contained 0.1 M NaCl or KCl, 10 mM MgCI2, 50 mM histidine/glycerophosphate or 50 mM TrislMops. Aliquots were taken and filtered through Schleicher & Schiill BA85,0.45 pm, at appropriate time intervals. For both uptake and release of calci um and strontium, appropriate proteinfree blanks were tested by filtering protein-free solutions. The calcium-transport ratio was determined by monitoring the burst of dinitrophenol liberated following the addition of 3 - 10 nmol/ml calcium to the assay medium containing 0.1 M KCI, 0.1 M sucrose, 10 mM MgC12, 5 mM oxalate, 25 mM Tris/Mops or Tris/maleate, pH 7, and 0.3 mM DnpP. Reagents
Lutidine DnpP was synthesized by Mr. H. Gaukler according to [14] in our laboratories. A23187 was purchased from calbiochem GmbH, Frankfurt, FRG; other reagents were bought from Serva, Heidelberg, FRG, and Sigma, Deisenhofen, FRG.
RESULTS The dependence on calcium and strontium concentrations of the activity of the calcium transport enzyme was first analysed with A23 187-treated light sarcoplasmic reticulum vesicles using DnpP as substrate. The enzyme is activated by calcium in the same concentration range, 0.1 - 1.O pM, as that reported for calcium-dependent ATP hydrolysis 1131. Strontium activation was measured in the presence of 0.5 mM EGTA to suppress activation by contaminating calcium [8]. This is possible because the affinity of EGTA for strontium is lower by a factor of 1000 than that for calcium. The dissociation constant of strontium-EGTA was determined with an ion-sensitive electrode under our experimental conditions. The obtained value of 0.1 mM agrees quite well with the dissociation constant reported by Schwarzenbach et al. [I 51. On account of the great difference between the dissociation
0.03
a 1.0 10 0
02 Strontium (mM)
Strontium * 0 - ~ 0 rCclcium I O - ' ( ~ V )
Fig. 1. Strontium activation of' DnpP hydro1,vsis by sarcoplusmic reticulum vesicles. The assays contained 1 niM DnpP, 10 m M MgCIZ, 20 mM histidine/glycerophosphate, 0.1 M NaCI, 0.5 m M EGTA, 0.1 mgjmlvesicularprotein, 2 pM A23187; t = 20°C. (A) Dependence of strontium-activated DnpP hydrolysis on free strontium. (B) Hill plot of strontium-activated DnpP hydrolysis (A)and comparison of calcium-activated hydrolysis (M). A and A,,, are the actual and maximal enzyme activities, respectively 600 1
0.600 1.000
10.000 Calcium or Strontium (mM)
Fig. 2. Inhibition by strontium or calcium of DnpP hydrolysis of'sarcoplasmic reticulum vesicles. The assays contained 1 mM DnpP, 10 mM MgCIZ, 20 mM histidine/glycerophosphate ( A , 0) or 50 mM Tris/ Mops (A , O ) ,0.1 M NaC1,O.l mg/ml vesicular protein, 2 pM A23187 and the concentrations of calcium and strontium shown. Strontiumactivated DnpP hydrolysis was measured in the presence of 0.5 mM EGTA (A,A). Calcium-dependent DnpP hydrolysis (0.0).t = 20°C constant of calcium- and strontium-EGTA, EGTA complexes only very little of the added strontium while calcium contamination of the system is reduced below activating levels. As shown in Fig. 1 A and B, strontium activation occurs in the submillimolar range (K, = 0.3 mM) having a relatively flat slope resulting in a Hill coefficient of 1.2, which is markedly smaller than that obtained for calcium activation. Inhibition of the calcium-transport enzyme by calcium ion concentrations exceeding 0.1 mM is a characteristic feature of the enzyme. Fig. 2 shows that enzyme activity is suppressed by 50%)at a concentration of 1 mM and that its decline occurs within one order of magnitude with respect to concentration [16, 171. In contrast to calcium, 20 mM strontium does not significantly interfere with DnpP hydrolysis. Even in the presence of 30 mM strontium (not shown), enzyme activity is only partially suppressed. Yet the effect of high concentrations of strontium is difficult to judge because of almost unpredictable interference with the ion composition of the system. Fig. 2 further illustrates that the observed activity pattern does not depend on the nature of the pH buffer used in these experiments.
537 Table 1. Effect of different proton buffers on the DnpP hydrolysis of permeable and native surcoplasmic reticulum vesicles activated by calcium or strontium in Nu+-containing media. The assays contained 1 mM DnpP, 10 mM MgC12, 0.1 M NaCl, 0.2 mM CaCI2 or 3.0 mM SrC1, with 0.5 mM EGTA, the listed buffers and 0.02 mg/ml permeable light vesicles; t = 20°C. The rates are given as means f SEM; n = 6 Proton buffer
DnpPase activation by strontium of permeable vesicles
calcium of native vesicles
permeable vesicles
native vesicles
106.3 _+ 7.48 (n = 6)
550.5 _+ 8.7 (n = 8)
52.8 & 3.75 ( n = 6) 75.5 & 4.8 ( n = 7)
nmol . mg-' . min-' Tris/Mops (50 mM)
414.4 f 8.11 (n = 8)
Histidine/glycerophosphate (20 mM)
475.8
20.3 (n = 7)
283.5 k 16.6 ( n
=
7)
565.0 5 24.0 ( n
Tris/maleate (20 mM)
435.9 & 13.2 (n = 3)
264.5 k 16.04 (n
=
3)
-
The observation that high concentrations of calcium do, while those of strontium do not, produce measurable inhibition of DnpP hydrolysis of permeable vesicles, is in line with the enzymatic behavior of closed native vesicles. At calcium concentrations whch optimally activate the DnpP hydrolysis of calcium-permeable vesicles, the hydrolysis of closed vesicles reaches less than 20% of maximal activity, irrespective of the buffer composition of the media. Strontium-activated DnpP hydrolysis by closed vesicles behaves quite differently with respect to extent and buffer sensitivity of trans-inhibition. In the presence of histidine/glycerophosphate or of Tris/maleate the activity of closed vesicles is only 40% lower than that of calcium-permeable vesicles. In contrast, when the system is buffered with Tris/Mops or sodium/Mops, the activity of the closed vesicles drops to 25% of that of permeable control preparations (Table 1). The data given in Table 1 show that buffer composition affects the extent of trans-inhibition by calcium much less than that produced by strontium. This finding suggests that strontium storage produces strong inhibition only in the presence of Tris/Mops, while calcium storage reaches inhibitory concentrations with all applied buffers. The marked difference in the effects of the respective buffers on enzyme activity of the closed vesicles indicates that the buffers might interfere with the ion-storing capacity of the vesicle preparations. Uptake experiments performed under corresponding conditions furnished the expected results, and in addition revealed that the enhancement of ion uptake by Mops markedly depends on whether sodium or potassium ions are present as monovalent cations. In the presence of sodium, strontium uptake energized by DnpP as well as by ATP is strongly enhanced in the presence of Mops, as compared to histidine or Tris/maleate. If potassium is the prevalent monovalent ion, uptake activation produced by Mops is less pronounced (Table 2). In contrast, calcium uptake is less affected by medium composition with respect to buffers and monovalent cations. Calcium uptake in Mops-buffered media is twofold higher than in histidine-buffered or Tris/maleate-buffered media, irrespective of the presence of sodium or potassium. The uptake transients observed for ATP-supported uptake do not interfere with our reasoning (not shown). Similar transients were reported by Sorenson and de Meis [18] in Tris/maleate-buffered media at pH 6.0. The large effects which proton buffers and ions exert on strontium and calcium-storing capacity cannot be related to alterations in the activity of the forward-running ion pump, as revealed by small variations in enzyme activity
=
11)
-
Table 2. Dependence of ion storage on energy supply, monovalent cations and proton buffers. Ion storage was determined after an uptake period of 15 min in the assays given in Materials and Methods. The assays containing 1 mM DnpP or 2 mM ATP, 10 mM MgCI,, the listed buffers, 0.1 M NaCl or 0.1 M KCl, 20 pM 45Ca or "Sr and 0.05 mg/ml native light vesicles; t = 20°C Ion stored Proton buffer
Energy supplied by DnpP with
Na+
ATP with K'
Na'
K+
nmol/mg Strontium Histidine/ glycerophosphate (20 mM) Mops (50 mM) Calcium
10 60-
70
15 5
Histidine/ glycerophosphate 60- 70 60 (20 mM) Mops(50mM) 100-110 120
30 50-90
10 20-35
60
50
120
100
of leaky vesicles under corresponding conditions (Table 1). Therefore, it appears likely that buffers and ions might inhibit calcium efflux. To measure calcium efflux, the vesicles were at first actively loaded for 10 - 20 min with strontium or calcium using DnpP as the energy donor. Subsequently, release was initiated by diluting 1 vol. uptake medium with 3 vol. of the corresponding release media containing sodium as the main cation. The results illustrated in Fig. 3A and B show that calcium and strontium efflux occur much faster in histidine/ glycerol than in Mops-buffered media. The same is true, to a lesser extent, if sodium as the main cation in the release medium is exchanged for potassium (Table 3).
DISCUSSION The results presented in this study show that the pathway for calcium and strontium efflux through the membranes of light sarcoplasmic reticulum vesicles is specifically blocked when Mops is used as the proton buffer instead of other buffers, e. g. histidine/glycerophosphate or Tris/maleate. Calcium-releasing channels, present in heavy sarcoplasmic reticulum vesicles, are absent in the light preparations used and are blocked by the presence of 10 mM magnesium [lo].
538 A
B
storage are enhanced by the buffer. Like passive calcium or strontium efflux, the enhancement of ion uptake by Mops as 0compared to histidine/glycerophosphate depends on whether I the uptake medium contains mainly sodium or potassium E ions. In general, calcium uptake and release are considerably 0 0 80 E less affected by Mops than the corresponding strontium move60.v ments. Sodium in the medium augments the effect of Mops E P A on storage and release of both ions. 40.The observed rates of passive efflux in the absence as well 5+. ._ E as in the presence of Mops (Fig. 3, Table 3) are at variance 2 20.with the high rates of calcium or strontium-supported DnpP A .-A -' hydrolysis persisting after ion uptake (Table 1) has reached 10 15 20 25 30 steady state. Since this activity has been shown to be directly 10 15 20 25 30 Time (mi.) related to calcium or strontium turnover 16, 81, we have to T'me ( m i l ) Fig. 3. Calcium or strontium release modified by medium buffers. The assume that during active accumulation in the absence of conditions for measuring the release are given in Materials and precipitating ions that an ion efflux must occur which is much Methods. The media in (A) and (B) contained 0.1 M NaCl and (A) faster than the observed rates of passive release. This rapid 20 mM histidine/glycerophosphate or ( 0 )50 mM Tris/Mops, respec- ion efflux is most likely mediated by the active pump itself. tively. (A) Calcium release; (B) strontium release Such an ion-exit pathway could prevent overloading of the sarcoplasmic reticulum if the amount of ions offered for storage exceeds the physiological storage capacity which Tdbk 3. EJfect of' different proton buffers on the rate of calcium or might be limited by the amount of binding proteins 1231 and strontium releu.c.e.from actively loaded sarcoplasmic reticulum vesicles by the level of inorganic phosphate [16, 241. The existence of in sodium- or pota,r.riiim-contailzin~media. The composition of the such a pump-related efflux in sarcoplasmic reticulum memassays for calcium or strontium loading is described in Materials and Methods. The release media contained 0.1 M NaCl or 0.1 M KCI, branes has been proposed by Waas and Hasselbach [6] and 10 mM MgCI2 and the listed buffers. Release was initiated by diluting discussed in relation to pump efficiency by Johnson et al. [7]. 1 vol. uptake medium with 3 vol. release medium It remains to be established if the slow passive efflux is also mediated by the pump but in a non-energized mode. Ion stored Proton buffer Initial rate of ion release An efflux mediated by the active pump would lead to a fast turnover of the energy donors ATP or DnpP at the cessation of K' Na net calcium uptake, and thus to continuous energy dissipation. Yet, in the case of calcium storage, this situation does not nmol. mg-' . min-' occur because the transport enzyme is inhibited when the calcium concentration at the lumenal surface reaches 3 Strontium Histidine glyccrophosphate 5 mM, which is rapidly reached inside native vesicles in the (20 mM) 40 31 absence of precipitating ions [17]. Mops (50 mM) 15 30 The reasoning above can also be applied to account for the behavior of the transport system during strontium acCalcium Hist idinc glycerophosphdte cumulation and its strong modulation by Mops. In contrast (20 m M ) 20 45 to calcium, strontium does not inhibit the enzymatic activity Mops (50 mM) 10 25 of the pump enzyme, even in the presence of 20- 30 mM, which is at least 10 times higher then the inhibitory concentration of calcium. Hence strontium, in contrast to calcium accumulation, is not truncated by pump inhibition. Evidently Therefore they are not part of the calcium-efflux pathway the lumenal binding sites of the enzyme related to pump inhiaffected by Mops. Mops affects neither the calcium- nor the bition have a much lower affinity for strontium than for strontium-activated hydrolysis of ATP or DnpP. Further- calcium. Since in the absence of Mops the proceeding active more, it has repeatedly been shown that Mops does not inter- strontium uptake only results in a small steady-state strontium fere with ATP-supported calcium uptake in the presence of the load (compare Table 2), we have to assume that strontium, calcium precipitating agent, oxalate. ATP-supported-calcium- although its concentration remains quite low, can rapidly be uptake measurements in the presence of Mops [19,20] yielded carried outwards, as already shown by Mermier and rates which fully agree with those observed in the presence Hasselbach [8]. Mops evidently interferes with this reaction. of other buffers [I, 211. Furthermore, calcium uptake in the The marked inhibition of DnpP hydrolysis by closed native presence of oxalate supported by DnpP, which was first de- vesicles can only be produced by a significant increase of scribed by Rossi et al. [22], furnished the same transport ratio internal strontium exceeding 30 mM. Strontium concen(calcium taken up)/DnpP hydrolysed) of 1.4 in media buffered trations inside the vesicles, in the order of 0.3 M, have been with either Mops or Trislmaleate. Thus, we can presume that reported for ATP-dependent strontium storage by Mermier the coupling between substrate hydrolysis and ion translo- and Hasselbach [8]. These concentrations could be reached cation is not differently affected by the buffers. under our experimental conditions selected for measuring enIn the absence of oxalate, where the internal ion concen- zyme activity if only 1% of the added strontium is stored by trations rapidly reach quite high values, the interference of the vesicles. Mops with ion storage becomes manifest, whereby the storage As already mentioned, the effect of Mops on calcium acof strontium is affected to a much larger extent than that of cumulation, and calcium activated steady-state DnpP hycalcium. The retardation of passive calcium efflux produced drolysis by native vesicles, is much smaller than that on the by Mops is in line with the finding that calcium and strontium corresponding reaction with strontium. This can most likely 1007
4
20
\
'\'
y1
c
v)
+
539 be related to the low rate of calcium turnover resulting from severe trans-inhibition. A further compensatory reduction of the rate of calcium efflux would only lead to a small increase of the lumenal free-calcium concentration, and as a consequence to a small increment of bound calcium. As for the role of monovalent cations in the effect of Mops, specific differences between calcium and strontium storage have been observed. The enhancement of calcium storage by Mops is the same in the presence of sodium and potassium (compare Table 2), and does not depend on the activity-yielding substrate. In contrast, Mops distinctly enhances strontium uptake only if sodium is the main monovalent cation. This indicates that the efflux of calcium and strontium through the active pump depends in a rather complex manner on the interaction of the transport enzyme with various ionic components, buffers, monovalent and divalent ions. We are indebted to Mr. A. Pfandke for his help in preparing the sarcoplasmic reticulum vesicles.
REFERENCES 1. Makinose, M. & Hasselbach, W. (1965) Biochem. Z. 343, 360382. 2. Weber, A,, Herz, R. & Reiss, I. (1966) Biochem. Z. 345, 329369. 3. Hasselbach, W. (1966) Ann. N. Y . Acad. Sci. 137, 1041- 1048. 4. Hasselbach, W. & Oetliker, H. (1983) Annu. Rev. Physiol. 45, 125- 139. 5. Makinose, M. & Hasselbach, W. (1971) FEBS Lett. 12, 271272.
6. Waas, W. & Hasselbach, W. (1981) Enr. J . Biochem. 116, 601 608. 7. Johnson, E. A., Tanford, Ch. & Reynolds, J. A. (1985) Proc. Nut1 Acad. Sdi. U S A 82, 5352 - 5356. 8. Mermier, P. & Hasselbach, W. (1976) Eur. J. Biochem. 69, 7986. 9. Guimares-Motta, H., Sande-Lemos, M. P. & de Meis L. (1984) J . Biol. Chem. 259, 8699-8705. 10. Meissner, G. (1984) J . Bid. Chem. 259, 2365 - 2374. 11. Su, J. & Hasselbach, W. (1984) Pfliigers Arch. 400, 14-21. 12. de Meis, L. & Hasselbach, W. (1971) J . Biol. Chem. 246, 47594163. 13. Hasselbach, W. (1988) Z . Nuturforsch. 43c, 929-937. 14. Ramirez, F. & Maracek, J. F. (1978) Synthesis 5, 601 -603. 15. Schwarzenbach, G. (1960) Die komplexometrische Titration, Ferdinand Enke Verlag, Stuttgart. 16. Inesi, G. & de Meis, L. (1989) J . Biol. Chem. 264, 5929-5936. 17. Hasselbach. W. (1976) Molecular bnsis of motility (Heilmeyer, L. M. G. et al., eds) pp. 81 -92, Springer V. Berlin, Heidelberg. 18. Sorenson, M. M. & de Meis, L. (1917) Biochim. Biophys. Actu 465,210-223. 19. de Meis, L. & Sorenson, M. M. (1989) Biochim. Biophys. Actu 984, 313 - 378. 20. Gafni, A. & Boyer, P. D. (1985) Proc. Natl Acad. Sci. USA 82, 89-101. 21. Hasselbach, W. & Migala, A. (1985) Z. Naturforsch. 40c, 571 575. 22. Rossi, B., Leone, F., Gacho, Ch. & Lazdunski, J. M. (1979) J . Biol. Chem. 254, 2302 - 2307. 23. MacLennan, D. H., Ostwald, T. J. & Stewart, P. S. (1973) Ann. N . Y. Acad. Sci. 227,527- 536. 24. Fassold, E. & Hasselbach, W. (1986) Eur. J . Biochem. 154, 714.
