ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 193, No. 1, March, pp. 22-27, 1979
Alkalinization
within Sarcoplasmic Uptake of Calcium VfTOR
Centro
de Biologia
Celular, Received
Departamento July
Reticulum Ions
during
the
M. C. MADEIRA de Zoologia,
28, 1978; revised
Universidade September
de Coimbra,
Portugal
31, 1978
The pH indicator, bromothymol blue, was incorporated into sarcoplasmic reticulum vesicles which bind more than 90% of the total added dye. The sequestered dye does not respond to changes in external pH upon addition of acid to the medium, since the decrease of absorbance at 616 nm is very slow. The absorbance of sequestered dye at 616 nm increases suddenly after triggering the transport of CaZ+ by ATP at a rate much higher than that of Ca*+ uptake, and declines when Ca *+ has been accumulated. When the uptake of CaZ+ is followed in the presence of oxalate, the absorbance of the indicator declines after the first phase of Ca*+ uptake. The results suggest that a transient alkalinization occurs rapidly inside the vesicles and reflects the formation of a transmembrane proton gradient responsible for sustaining the CaZ+ transport.
suggested the formation of a transmembrane proton gradient on the basis of the spectrometric behaviors of the probes, 9amino-6-chloro-2-methoxyacridine and atebrin (9). The observed events associated with the transport of Ca2+ triggered by ATP, occur much faster than the transport of the cation and persist while Ca2+ is taken up by the vesicles. The present work provides evidence that a transient alkalinization occurs within the vesicles of sarcoplasmic reticulum as a consequence of Ca2+ transport triggered by ATP. The rate of this event is also faster than the rate of Ca2+ transport. The results obtained are consistent with our proposal of the formation of a transmembrane proton gradient during the early stages of Ca2+ uptake.
Energy transductions in biomembranes are often conveniently explained by a chemiosmotic coupling as proposed by Mitchell (1, 2). The chemiosmotic hypothesis explains biomembrane energy transductions by the establishment of transmembrane proton or electrical gradients (or both) as the primary events. The transmembrane gradients may be coupled to processes which require energy, namely, phosphorylation of ADP (1, 2) and transport of solutes (3, 4). Sarcoplasmic reticulum vesicles take up calcium ions at the expense of ATP hydrolysis by a mechanism whose details have been extensively characterized (5-8). This membrane system is formed by lipids and a single functional enzyme, the Ca2+-pump assembly. The simplicity of such a system makes it particularly attractive, as a native model system, for the study of the mechanism of cation transport. Although a great effort has been made to clarify the mechanism of Ca2+ transport little attention has been directed toward the primary events which precede the transport of Ca2+ and transduce the energy of ATP into osmotic energy of Ca2+ gradients. Recently, I have shown that protons are extruded before Ca2+ transport and have 0003-9861/79/030022-06$02.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS
AND
METHODS
Membranes of sarcoplasmic reticulum were obtained from rabbit muscles as described elsewhere (10). More than 80% of total membrane protein was Ca*+-ATPase,’ as estimated by densitometry of polyacrilamide electrophoretogrdms (11). The preparations were used within 2 days after preparation. 1 Abbreviation phosphatase. 22
used:
ATPase,
adenosinetri-
ALKALINIZATION
DURING
SARCOPLASMIC
The binding of bromothymol blue was studied in membranes (5 mg of protein) incubated at room temperature for 5 min in 10 ml of basic reaction medium (50 mM KCl, 5 mM MgC&, 20 mM Trismaleate, pH 6.9) supplemented with varying concentrations of the indicator. The suspensions were then centrifuged at 105g for 45 min, and the absorbance values of the supernatants were read at 616 nm, after bringing the pH to 9.0 with NaOH. The amounts of bound bromothymol blue were estimated taking an empirical eGLBnm(pHy.,j)of 56,675 M-l cm-‘. The changes of absorbance during the transport of Ca’+ were monitored by dual wavelength spectrophotometry (616-500 nm) in an Aminco DW-2 system. The membranes were suspended in 2.5 ml of basic reaction media containing 8 pM bromothymol blue, CaCl,, and other substances as stated in the figure legends. The reactions were initiated by rapid mixing of Mg-ATP (0.2 mM final concentration) delivered from an Aminco mixing device catalog No. B3-65085. Rapid kinetic experiments were performed on an Aminco-Morrow stopped-flow apparatus. Syringe A contained a suspension of vesicles (0.8 mgiml) in basic medium supplemented with 8 pM bromothymol blue and 80 pM CaCl,, and syringe B contained 0.4 mM ATP in basic medium. The uptake of Ca’+ was monitored with a Ca2+ electrode as described previously (9, 12) in media containing membranes and CaCl, as stated in the figure legends. The reactions were initiated by adding MgATP. Care was taken to maintain the Ca2+ concentration above lo-” M, since the electrode responds satisfactorily only above this concentration. The Ca*+ uptake in the presence of oxalate was followed continuously by monitoring the production of protons arising from the ATP hydrolysis (see Ref. 9 for rationale).
RETICULUM
Ca”+ UPTAKE
23
FIG. 1. Binding of bromothymol blue by sarcoplasmic reticulum vesicles.
vesicles (9), it would be difficult to discriminate from the spectrometric information, the signals due to pH changes within the vesicles. Additionally, it was shown in chloroplasts that the absorbance changes of neutral red may reflect accumulation of the dye inside the organelles during proton pumping (14). Therefore, I preferred the use of bromothymol blue to monitor pH changes, since it binds to the membranes with high affinity, and it has been used successfully in a variety of energy-transducing membranes (13, 15-17). It is shown in Fig. 2 that the bound dye does not respond to the external pH of the medium as estimated by the change in absorbance at 616-500 nm upon addition of HCl to the suspensions. The reference wavelength, 500 nm, is the isobestic point of the pH-dependent spectra (Fig. 3). Addition of an amount of acid sufficient to cause RESULTS AND DISCUSSION a change in pH from 6.9 to 6.76 induces a Bromothymol blue added to membrane sus- very limited decrease in absorbance of the pensions binds to the membranes with high dye in membrane suspensions. This limited affinity, since about 90% of the total added but rapid effect probably reflects the beremains bound (Fig. l), when the total havior of the small amount of free external added dye does not exceed 20 nmollmg of dye in equilibrium with the bound dye. It protein. A ratio of indicator to protein lower appears that the absorbance of the bound than this value was used in the experiments dye decreases very slowly after addition of described below, since it is of interest to acid to the medium which suggests that follow the behavior of sequestered dye bound bromothymol blue is not exposed to rather than the free dye. The other pH indithe external medium, since the external cator, neutral red, often used also to moniproton concentration is not readily sensed. tor internal pH of various organelles Therefore, the indicator is presumably lo(13-15) proved to be less useful in sarco- cated in the vicinity of the inner vesicular plasmic reticulum, since about 50% of the space, in a domain not accessible from the total added dye remains free in the external outside. Furthermore, the very slow absormedium. Since protons are produced during bance decrease of the bound dye provides Ca2+ uptake by sarcoplasmic reticulum further evidence that the membranes of
24
VfTOR M. C. MADEIRA
bance (616-500 nm) of bound bromothymol blue, indicating that a transient alkalinization occurs within the vesicles during the transport of Ca2+ triggered by ATP. It was shown in previous work (9) that protons are ejected from the vesicles during the uptake of Ca2+. Therefore, if the bound indicator remained near the external surface of the vesicles, a protonation of the dye would induce a decrease in absorbance at 616 nm, ‘lo since the pK of bromothymol blue is 7.0 under the experimental conditions used herein. Thus, I shall interpret the increase of absorbance as a consequence of an alkalinization occuring within the vesicles or near the inner surface of the membrane, itself a consequence of extrusion of protons during the uptake of Ca2+. It then appears FIG. 2. Effect of acid addition on the absorbance of that bromothymol blue indicates pH changes bromothymol blue. HCl was added in amounts suf- in the sites where Ca2+ is accumulated, ficient to cause a pH drop from 6.90 to 6.76. The effect of acid addition is very limited when vesicles of i.e., the inside of the vesicles. Very similar sarcoplasmic reticulum (2.5 mg of protein in 2.5 ml conclusions were achieved for the behavior blue used as pH indicator in of medium) were present (trace to the right). Note also of bromothymol during the uptake of Ca2+ that there is a slow decrease in absorbance after the mitochondria (16, 17). Furthermore, it appears that the initial limited but rapid effect. The basic reaction media (with or without vesicles) were supplemented indicator has the general property of bindwith 8 pM bromothymol blue and 40 PM CaCl,. The ex- ing in the vicinity of the inner surface of periments were carried out by dual wavelength spec- other organelles, including tilakoids of trophotometry taking 616 and 500 nm as sampling and chloroplasts (13) and bacterial chromatoreference wavelengths, respectively. Control experiphores (15). ments with disrupting agents of membranes, viz., deThe alkalinization of sarcoplasmic reticutergents, were inconclusive since the detergents lum monitored by sequestered bromoquench the absorbance of bromothymol blue. thymol blue is transient (Fig. 4) and lasts only the time required for the uptake of sarcoplasmic reticulum are quite imperCa2+. When the total amount of Ca2+ added meable to protons. is below the total capacity of the vesicles The results summarized in Fig. 4 show (100 to 150 nmol/mg of protein), the absorthat there is a transient increase of absor- bance signal rises rapidly after ATP addi-
02
c
350
SW
A (nm)
550
650
loo
FIG. 3. Absorbance spectra of bromothymol blue. The indicator (4 WM) was added to the basic reaction media containing 40 PM CaCl, and adjusted to the desired pH values. Spectra were recorded in a DW-2 Aminco spectrophotometer (split&am mode) at 3-nm band pass. The reference cell contained the medium without the indicator.
ALKALINIZATION
DURING
SARCOPLASMIC
RETICULUM
Ca*+ UPTAKE
25
recycling of Ca2+ across the membranes maintains the operation of the pumping system, thus preventing the dissipation of the alkalinization in the sequestering space of bromothymol blue. The alkalinization triggered by addition of ATP is only observed in intact membranes. Membranes disintegrated by addition of detergent (Fig. 4) do not accumulate Ca2+ and do not show the typical transient alkalinization. Only a very small effect, proba 0-i ably resulting from an artifact, may be obP served sometimes (Fig. 4). Therefore, the -3 zotransient alkalinization only occurs in 5 10vesicles which are able to accumulate Ca2+. a The results summarized in Fig. 5 rein&SWforce the previous conclusion that an interitlo. nal alkalinization is required to maintain the ‘TRITON (OOtransport of Ca2+ and to sustain the Ca2+ gradients across the membranes. When FIG. 4. Absorbance changes of bound bromothymol blue during the uptake of Ca2+ by sarcoplasmic reticuoxalate is present, all the added Ca2+ is lum. The reaction media were supplemented with virtually taken up and precipitates inside vesicles (2.5 mg), bromothymol blue (8 PM), and CaCl, the vesicles in the form of calcium oxalate as indicated on the traces, in a total volume of 2.5 ml. (18). The precipitation renders calcium ions The numbers on the traces refer to the amounts of osmotically inactive, avoiding high Ca2+ added CaCl,. The labeling zero means that no CaCl, concentrations inside the vesicles and, was added to the medium in this particular experiment. hence, leaking of Ca2+ to the outside. It is The actual amount of Ca2+ (residual Ca*+) was about shown in Fig. 5 that alkalinization within 9 nmol, as determined by atomic absorption spectrothe vesicles begins to decrease at the end metry. The reactions were initiated by adding MgATP (500 nmol). The Ca*+ uptake profile for 400 nmol of the first phase of Ca2+ uptake (best of added CaCl, is also shown. The final concentration seen for 200 nmol of added Ca2+), preof Triton X-100, when added, was 0.06%. The dotted sumably because precipitation of calcium trace represents Ca2+ uptake after solubilization of the oxalate begins by this time (19, 20). Since membranes with Triton. Note that the response of the the Ca2+ gradient vanishes when precipitaCaZ+ electrode is fast enough to estimate rates of Ca*+ tion begins, the motive force which sustains uptake since a rapid deflection of the trace is seen as a the gradient, supposedly associated with consequence of Ca*+ release when Triton is added at the alkalinization process, dissipates and the end of Ca*+ uptake. thus the pH of the inner vesicular space assumes the values existing before the uptake tion, and it vanishes after a brief period. of Ca2+. The traces of Fig. 5, for high The extent of this period of time depends concentrations of added Ca2+, seem to on the amount of Ca2+ added and, thus, return to values below the baseline probon the time required for Ca2+ uptake. How- ably as a consequence of the light scatterever, when the amount of Ca2+ added ing effect due to the precipitation of masexceeds the capacity of the vesicles (about sive amounts of calcium oxalate. Some 100 nmoVmg of protein for preparation of artifactual signal resembling decrease in abFig. 4), the absorbance signal remains sorbance is expected since the reference high for long periods of time, indicating wavelength, 500 nm, is lower than the that permanent alkalinization is maintained sampling wavelength (616 nm). within the vesicles. In this last situation, The kinetics of the absorbance changes large gradients of Ca2+ across the memof sequestered indicator is shown in Fig. 6. branes favor continuous leaking out of cal- The stopped-flow trace shows that the maxicium ions which are again taken up. The mum absorbance signal develops in less than 1
26
VfTOR M. C. MADEIRA
FIG. 5. Absorbance changes of bromothymol blue during Ca*+ uptake in the presence of oxalate. The basic reaction media were supplemented with membranes (2.5 mg), 5 mM potassium oxalate, 8 f&M bromothymol blue, and CaCl, as indicated. The reactions were initiated by adding Mg-ATP (600 nmol). Note that the Ca2+ uptake profiles exhibited two distinct phases (best seen for 400 and 200 nmol CaCl,).
s, and more than 80% of the maximum occurs 0.5 s after triggering the Ca2+ uptake. The amount of Ca2+ accumulated in 1 s by the vesicles in the suspension is about 15% of the total capacity of the vesicles, and in 0.5 s this figure is as low as 8%. It should be noted that the total Ca2+ added to the suspensions (100 nmol/mg of protein) is fairly below the total capacity of these membranes which take up more than 150 nmol/mg of protein. Therefore, all the added
Ca2+ in conditions of the experiments of absorbance monitoring is easily taken up by the vesicles. The extent of alkalinization within the vesicles could not yet been estimated, since it has been difficult to make accurate calibrations of the changes in absorbance of the sequestered indicator in terms of internal pH alterations. However, some of the technical difficulties have been removed, so that experiments designed to equilibrate internal and external pH are now in progress to attempt the calibration of the absorbance changes of bromothymol blue within the vesicles of sarcoplasmic reticulum. Since the rate of alkalinization in the vesicles is much faster than the rate of Ca2+ uptake, it is suggested that alkalinization within the vesicles is a primary event in the process of Cal+ transport. The conclusions drawn in this work are in close agreement with those of a previous work (9) where I have shown that a priFIG. 6. Kinetics of absorbance changes of bromothymary proton ejection occurs during the mol blue during the uptake of Ca*+. The reactions transport of Ca2+ and suggested the formawere initiated by adding Mg-ATP (0.2 mM, final con- tion of a transmembrane proton gradient. centration). The basic reaction media were supple- Therefore, this work supports quite clearly mented with membranes (0.4 mg/ml), 4 pM bromothythe conclusion that a transmembrane proton mol blue, and 40 pM CaCl,. The trace shown below gradient is a primary event occurring durrepresents the uptake of Ca2+ in similar conditions as of Ca2+ in sarcoplasmic those described above, except, for technical reasons, ing the transport and is probably the motive force the final concentration of added CaCl, was 80 pM (see reticulum which sustains the transport. text for more details).
ALKALINIZATION
DURING
SARCOPLASMIC
ACKNOWLEDGMENTS I am indebted to Dr. M. Tully for correcting the English manuscript. This work was supported by a grant of I.N.I.C. (Portuguese Ministry of Education and Culture).
REFERENCES 1. MITCHELL, 2. 3. 4. 5. 6. 7.
P. (1966) Biol. Rev. Cambridge Phil. Sot. 41, 445-502. MITCHELL, P. (1972) J. Bioenerg. 3, 5-24. MITCHELL, P. (1973) FEBS Lett. 33, 267-274. TSUCHIYA, T., AND ROSEN, B. P. (1976) J. Biol. Chem. 251, 962-967. WEBER, A. (1966) Curr. Top. Bioenerg. 1, 203254. HASSELBACH, W., AND MAKINOSE, M. (1962) Biothem. Biophys. Res. Commun. 7, 132-136. MARTONOSI, A. (19’72) in Current Topics in Bio-
energetics (Bronner, F., and Kleinzeller, A., eds.), Vol. 3, pp. 83-197, Academic Press, New York. 8. TADA, M., YAMAMOM, T., AND TONOMURA, Y. (1978) Physiol. Rev. 58, l-79.
RETICULUM
Gas+ UPTAKE
27
9. MADEIRA, V. M. C. (1978) Arch. Biochem. Biophys. 185, 316-325. 10. CARVALHO, A. P., AND MOTA, A. (1971) Arch. Biochem. Biophys. 142, 201-212. 11. MADEIRA, V. M. C. (1977) Biochim. Biophys. Acta 464, 583-588. 12. MADEIRA, V. M. C. (1975) Biochem. Biophys. Res. Commun. 64, 870-876. 13. LYNN, W. S. (1968) J. Biol. Chem. 243, 10601064. 14. PICK, U., AND AVRON, M. (1976) FEBS Lett. 65, 348-353. 15. NISHI, N., SAKATA-SOGAWA, K., SOE, G., AND YAMASHITA, J. (1977) J. Biochem. (Tokyo) 82,
1267- 1279. 16. CHANCE, B., ANDMELA, M. (1966)J. Biol. Chem. 241, 4588-4599. 17. GHOSH, A. K., AND CHANCE, B. (1970) Arch. Biochem. Biophys. 138, 483-492. 18. HASSELBACH, W. (1964) Progr. Biophys. 14, 167-222. 19. ENTMAN, M. L., SNOW, T. R., FREED, D., AND SCHWARTZ, A. (1973) J. Biol. Chem. 248, 7762-7772. 20. MERMIER, P., AND HASSELBACH, W. (1976) Eur. J. Biochem. 64, 613-620.
Alkalinization
within Sarcoplasmic Uptake of Calcium VfTOR
Centro
de Biologia
Celular, Received
Departamento July
Reticulum Ions
during
the
M. C. MADEIRA de Zoologia,
28, 1978; revised
Universidade September
de Coimbra,
Portugal
31, 1978
The pH indicator, bromothymol blue, was incorporated into sarcoplasmic reticulum vesicles which bind more than 90% of the total added dye. The sequestered dye does not respond to changes in external pH upon addition of acid to the medium, since the decrease of absorbance at 616 nm is very slow. The absorbance of sequestered dye at 616 nm increases suddenly after triggering the transport of CaZ+ by ATP at a rate much higher than that of Ca*+ uptake, and declines when Ca *+ has been accumulated. When the uptake of CaZ+ is followed in the presence of oxalate, the absorbance of the indicator declines after the first phase of Ca*+ uptake. The results suggest that a transient alkalinization occurs rapidly inside the vesicles and reflects the formation of a transmembrane proton gradient responsible for sustaining the CaZ+ transport.
suggested the formation of a transmembrane proton gradient on the basis of the spectrometric behaviors of the probes, 9amino-6-chloro-2-methoxyacridine and atebrin (9). The observed events associated with the transport of Ca2+ triggered by ATP, occur much faster than the transport of the cation and persist while Ca2+ is taken up by the vesicles. The present work provides evidence that a transient alkalinization occurs within the vesicles of sarcoplasmic reticulum as a consequence of Ca2+ transport triggered by ATP. The rate of this event is also faster than the rate of Ca2+ transport. The results obtained are consistent with our proposal of the formation of a transmembrane proton gradient during the early stages of Ca2+ uptake.
Energy transductions in biomembranes are often conveniently explained by a chemiosmotic coupling as proposed by Mitchell (1, 2). The chemiosmotic hypothesis explains biomembrane energy transductions by the establishment of transmembrane proton or electrical gradients (or both) as the primary events. The transmembrane gradients may be coupled to processes which require energy, namely, phosphorylation of ADP (1, 2) and transport of solutes (3, 4). Sarcoplasmic reticulum vesicles take up calcium ions at the expense of ATP hydrolysis by a mechanism whose details have been extensively characterized (5-8). This membrane system is formed by lipids and a single functional enzyme, the Ca2+-pump assembly. The simplicity of such a system makes it particularly attractive, as a native model system, for the study of the mechanism of cation transport. Although a great effort has been made to clarify the mechanism of Ca2+ transport little attention has been directed toward the primary events which precede the transport of Ca2+ and transduce the energy of ATP into osmotic energy of Ca2+ gradients. Recently, I have shown that protons are extruded before Ca2+ transport and have 0003-9861/79/030022-06$02.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS
AND
METHODS
Membranes of sarcoplasmic reticulum were obtained from rabbit muscles as described elsewhere (10). More than 80% of total membrane protein was Ca*+-ATPase,’ as estimated by densitometry of polyacrilamide electrophoretogrdms (11). The preparations were used within 2 days after preparation. 1 Abbreviation phosphatase. 22
used:
ATPase,
adenosinetri-
ALKALINIZATION
DURING
SARCOPLASMIC
The binding of bromothymol blue was studied in membranes (5 mg of protein) incubated at room temperature for 5 min in 10 ml of basic reaction medium (50 mM KCl, 5 mM MgC&, 20 mM Trismaleate, pH 6.9) supplemented with varying concentrations of the indicator. The suspensions were then centrifuged at 105g for 45 min, and the absorbance values of the supernatants were read at 616 nm, after bringing the pH to 9.0 with NaOH. The amounts of bound bromothymol blue were estimated taking an empirical eGLBnm(pHy.,j)of 56,675 M-l cm-‘. The changes of absorbance during the transport of Ca’+ were monitored by dual wavelength spectrophotometry (616-500 nm) in an Aminco DW-2 system. The membranes were suspended in 2.5 ml of basic reaction media containing 8 pM bromothymol blue, CaCl,, and other substances as stated in the figure legends. The reactions were initiated by rapid mixing of Mg-ATP (0.2 mM final concentration) delivered from an Aminco mixing device catalog No. B3-65085. Rapid kinetic experiments were performed on an Aminco-Morrow stopped-flow apparatus. Syringe A contained a suspension of vesicles (0.8 mgiml) in basic medium supplemented with 8 pM bromothymol blue and 80 pM CaCl,, and syringe B contained 0.4 mM ATP in basic medium. The uptake of Ca’+ was monitored with a Ca2+ electrode as described previously (9, 12) in media containing membranes and CaCl, as stated in the figure legends. The reactions were initiated by adding MgATP. Care was taken to maintain the Ca2+ concentration above lo-” M, since the electrode responds satisfactorily only above this concentration. The Ca*+ uptake in the presence of oxalate was followed continuously by monitoring the production of protons arising from the ATP hydrolysis (see Ref. 9 for rationale).
RETICULUM
Ca”+ UPTAKE
23
FIG. 1. Binding of bromothymol blue by sarcoplasmic reticulum vesicles.
vesicles (9), it would be difficult to discriminate from the spectrometric information, the signals due to pH changes within the vesicles. Additionally, it was shown in chloroplasts that the absorbance changes of neutral red may reflect accumulation of the dye inside the organelles during proton pumping (14). Therefore, I preferred the use of bromothymol blue to monitor pH changes, since it binds to the membranes with high affinity, and it has been used successfully in a variety of energy-transducing membranes (13, 15-17). It is shown in Fig. 2 that the bound dye does not respond to the external pH of the medium as estimated by the change in absorbance at 616-500 nm upon addition of HCl to the suspensions. The reference wavelength, 500 nm, is the isobestic point of the pH-dependent spectra (Fig. 3). Addition of an amount of acid sufficient to cause RESULTS AND DISCUSSION a change in pH from 6.9 to 6.76 induces a Bromothymol blue added to membrane sus- very limited decrease in absorbance of the pensions binds to the membranes with high dye in membrane suspensions. This limited affinity, since about 90% of the total added but rapid effect probably reflects the beremains bound (Fig. l), when the total havior of the small amount of free external added dye does not exceed 20 nmollmg of dye in equilibrium with the bound dye. It protein. A ratio of indicator to protein lower appears that the absorbance of the bound than this value was used in the experiments dye decreases very slowly after addition of described below, since it is of interest to acid to the medium which suggests that follow the behavior of sequestered dye bound bromothymol blue is not exposed to rather than the free dye. The other pH indithe external medium, since the external cator, neutral red, often used also to moniproton concentration is not readily sensed. tor internal pH of various organelles Therefore, the indicator is presumably lo(13-15) proved to be less useful in sarco- cated in the vicinity of the inner vesicular plasmic reticulum, since about 50% of the space, in a domain not accessible from the total added dye remains free in the external outside. Furthermore, the very slow absormedium. Since protons are produced during bance decrease of the bound dye provides Ca2+ uptake by sarcoplasmic reticulum further evidence that the membranes of
24
VfTOR M. C. MADEIRA
bance (616-500 nm) of bound bromothymol blue, indicating that a transient alkalinization occurs within the vesicles during the transport of Ca2+ triggered by ATP. It was shown in previous work (9) that protons are ejected from the vesicles during the uptake of Ca2+. Therefore, if the bound indicator remained near the external surface of the vesicles, a protonation of the dye would induce a decrease in absorbance at 616 nm, ‘lo since the pK of bromothymol blue is 7.0 under the experimental conditions used herein. Thus, I shall interpret the increase of absorbance as a consequence of an alkalinization occuring within the vesicles or near the inner surface of the membrane, itself a consequence of extrusion of protons during the uptake of Ca2+. It then appears FIG. 2. Effect of acid addition on the absorbance of that bromothymol blue indicates pH changes bromothymol blue. HCl was added in amounts suf- in the sites where Ca2+ is accumulated, ficient to cause a pH drop from 6.90 to 6.76. The effect of acid addition is very limited when vesicles of i.e., the inside of the vesicles. Very similar sarcoplasmic reticulum (2.5 mg of protein in 2.5 ml conclusions were achieved for the behavior blue used as pH indicator in of medium) were present (trace to the right). Note also of bromothymol during the uptake of Ca2+ that there is a slow decrease in absorbance after the mitochondria (16, 17). Furthermore, it appears that the initial limited but rapid effect. The basic reaction media (with or without vesicles) were supplemented indicator has the general property of bindwith 8 pM bromothymol blue and 40 PM CaCl,. The ex- ing in the vicinity of the inner surface of periments were carried out by dual wavelength spec- other organelles, including tilakoids of trophotometry taking 616 and 500 nm as sampling and chloroplasts (13) and bacterial chromatoreference wavelengths, respectively. Control experiphores (15). ments with disrupting agents of membranes, viz., deThe alkalinization of sarcoplasmic reticutergents, were inconclusive since the detergents lum monitored by sequestered bromoquench the absorbance of bromothymol blue. thymol blue is transient (Fig. 4) and lasts only the time required for the uptake of sarcoplasmic reticulum are quite imperCa2+. When the total amount of Ca2+ added meable to protons. is below the total capacity of the vesicles The results summarized in Fig. 4 show (100 to 150 nmol/mg of protein), the absorthat there is a transient increase of absor- bance signal rises rapidly after ATP addi-
02
c
350
SW
A (nm)
550
650
loo
FIG. 3. Absorbance spectra of bromothymol blue. The indicator (4 WM) was added to the basic reaction media containing 40 PM CaCl, and adjusted to the desired pH values. Spectra were recorded in a DW-2 Aminco spectrophotometer (split&am mode) at 3-nm band pass. The reference cell contained the medium without the indicator.
ALKALINIZATION
DURING
SARCOPLASMIC
RETICULUM
Ca*+ UPTAKE
25
recycling of Ca2+ across the membranes maintains the operation of the pumping system, thus preventing the dissipation of the alkalinization in the sequestering space of bromothymol blue. The alkalinization triggered by addition of ATP is only observed in intact membranes. Membranes disintegrated by addition of detergent (Fig. 4) do not accumulate Ca2+ and do not show the typical transient alkalinization. Only a very small effect, proba 0-i ably resulting from an artifact, may be obP served sometimes (Fig. 4). Therefore, the -3 zotransient alkalinization only occurs in 5 10vesicles which are able to accumulate Ca2+. a The results summarized in Fig. 5 rein&SWforce the previous conclusion that an interitlo. nal alkalinization is required to maintain the ‘TRITON (OOtransport of Ca2+ and to sustain the Ca2+ gradients across the membranes. When FIG. 4. Absorbance changes of bound bromothymol blue during the uptake of Ca2+ by sarcoplasmic reticuoxalate is present, all the added Ca2+ is lum. The reaction media were supplemented with virtually taken up and precipitates inside vesicles (2.5 mg), bromothymol blue (8 PM), and CaCl, the vesicles in the form of calcium oxalate as indicated on the traces, in a total volume of 2.5 ml. (18). The precipitation renders calcium ions The numbers on the traces refer to the amounts of osmotically inactive, avoiding high Ca2+ added CaCl,. The labeling zero means that no CaCl, concentrations inside the vesicles and, was added to the medium in this particular experiment. hence, leaking of Ca2+ to the outside. It is The actual amount of Ca2+ (residual Ca*+) was about shown in Fig. 5 that alkalinization within 9 nmol, as determined by atomic absorption spectrothe vesicles begins to decrease at the end metry. The reactions were initiated by adding MgATP (500 nmol). The Ca*+ uptake profile for 400 nmol of the first phase of Ca2+ uptake (best of added CaCl, is also shown. The final concentration seen for 200 nmol of added Ca2+), preof Triton X-100, when added, was 0.06%. The dotted sumably because precipitation of calcium trace represents Ca2+ uptake after solubilization of the oxalate begins by this time (19, 20). Since membranes with Triton. Note that the response of the the Ca2+ gradient vanishes when precipitaCaZ+ electrode is fast enough to estimate rates of Ca*+ tion begins, the motive force which sustains uptake since a rapid deflection of the trace is seen as a the gradient, supposedly associated with consequence of Ca*+ release when Triton is added at the alkalinization process, dissipates and the end of Ca*+ uptake. thus the pH of the inner vesicular space assumes the values existing before the uptake tion, and it vanishes after a brief period. of Ca2+. The traces of Fig. 5, for high The extent of this period of time depends concentrations of added Ca2+, seem to on the amount of Ca2+ added and, thus, return to values below the baseline probon the time required for Ca2+ uptake. How- ably as a consequence of the light scatterever, when the amount of Ca2+ added ing effect due to the precipitation of masexceeds the capacity of the vesicles (about sive amounts of calcium oxalate. Some 100 nmoVmg of protein for preparation of artifactual signal resembling decrease in abFig. 4), the absorbance signal remains sorbance is expected since the reference high for long periods of time, indicating wavelength, 500 nm, is lower than the that permanent alkalinization is maintained sampling wavelength (616 nm). within the vesicles. In this last situation, The kinetics of the absorbance changes large gradients of Ca2+ across the memof sequestered indicator is shown in Fig. 6. branes favor continuous leaking out of cal- The stopped-flow trace shows that the maxicium ions which are again taken up. The mum absorbance signal develops in less than 1
26
VfTOR M. C. MADEIRA
FIG. 5. Absorbance changes of bromothymol blue during Ca*+ uptake in the presence of oxalate. The basic reaction media were supplemented with membranes (2.5 mg), 5 mM potassium oxalate, 8 f&M bromothymol blue, and CaCl, as indicated. The reactions were initiated by adding Mg-ATP (600 nmol). Note that the Ca2+ uptake profiles exhibited two distinct phases (best seen for 400 and 200 nmol CaCl,).
s, and more than 80% of the maximum occurs 0.5 s after triggering the Ca2+ uptake. The amount of Ca2+ accumulated in 1 s by the vesicles in the suspension is about 15% of the total capacity of the vesicles, and in 0.5 s this figure is as low as 8%. It should be noted that the total Ca2+ added to the suspensions (100 nmol/mg of protein) is fairly below the total capacity of these membranes which take up more than 150 nmol/mg of protein. Therefore, all the added
Ca2+ in conditions of the experiments of absorbance monitoring is easily taken up by the vesicles. The extent of alkalinization within the vesicles could not yet been estimated, since it has been difficult to make accurate calibrations of the changes in absorbance of the sequestered indicator in terms of internal pH alterations. However, some of the technical difficulties have been removed, so that experiments designed to equilibrate internal and external pH are now in progress to attempt the calibration of the absorbance changes of bromothymol blue within the vesicles of sarcoplasmic reticulum. Since the rate of alkalinization in the vesicles is much faster than the rate of Ca2+ uptake, it is suggested that alkalinization within the vesicles is a primary event in the process of Cal+ transport. The conclusions drawn in this work are in close agreement with those of a previous work (9) where I have shown that a priFIG. 6. Kinetics of absorbance changes of bromothymary proton ejection occurs during the mol blue during the uptake of Ca*+. The reactions transport of Ca2+ and suggested the formawere initiated by adding Mg-ATP (0.2 mM, final con- tion of a transmembrane proton gradient. centration). The basic reaction media were supple- Therefore, this work supports quite clearly mented with membranes (0.4 mg/ml), 4 pM bromothythe conclusion that a transmembrane proton mol blue, and 40 pM CaCl,. The trace shown below gradient is a primary event occurring durrepresents the uptake of Ca2+ in similar conditions as of Ca2+ in sarcoplasmic those described above, except, for technical reasons, ing the transport and is probably the motive force the final concentration of added CaCl, was 80 pM (see reticulum which sustains the transport. text for more details).
ALKALINIZATION
DURING
SARCOPLASMIC
ACKNOWLEDGMENTS I am indebted to Dr. M. Tully for correcting the English manuscript. This work was supported by a grant of I.N.I.C. (Portuguese Ministry of Education and Culture).
REFERENCES 1. MITCHELL, 2. 3. 4. 5. 6. 7.
P. (1966) Biol. Rev. Cambridge Phil. Sot. 41, 445-502. MITCHELL, P. (1972) J. Bioenerg. 3, 5-24. MITCHELL, P. (1973) FEBS Lett. 33, 267-274. TSUCHIYA, T., AND ROSEN, B. P. (1976) J. Biol. Chem. 251, 962-967. WEBER, A. (1966) Curr. Top. Bioenerg. 1, 203254. HASSELBACH, W., AND MAKINOSE, M. (1962) Biothem. Biophys. Res. Commun. 7, 132-136. MARTONOSI, A. (19’72) in Current Topics in Bio-
energetics (Bronner, F., and Kleinzeller, A., eds.), Vol. 3, pp. 83-197, Academic Press, New York. 8. TADA, M., YAMAMOM, T., AND TONOMURA, Y. (1978) Physiol. Rev. 58, l-79.
RETICULUM
Gas+ UPTAKE
27
9. MADEIRA, V. M. C. (1978) Arch. Biochem. Biophys. 185, 316-325. 10. CARVALHO, A. P., AND MOTA, A. (1971) Arch. Biochem. Biophys. 142, 201-212. 11. MADEIRA, V. M. C. (1977) Biochim. Biophys. Acta 464, 583-588. 12. MADEIRA, V. M. C. (1975) Biochem. Biophys. Res. Commun. 64, 870-876. 13. LYNN, W. S. (1968) J. Biol. Chem. 243, 10601064. 14. PICK, U., AND AVRON, M. (1976) FEBS Lett. 65, 348-353. 15. NISHI, N., SAKATA-SOGAWA, K., SOE, G., AND YAMASHITA, J. (1977) J. Biochem. (Tokyo) 82,
1267- 1279. 16. CHANCE, B., ANDMELA, M. (1966)J. Biol. Chem. 241, 4588-4599. 17. GHOSH, A. K., AND CHANCE, B. (1970) Arch. Biochem. Biophys. 138, 483-492. 18. HASSELBACH, W. (1964) Progr. Biophys. 14, 167-222. 19. ENTMAN, M. L., SNOW, T. R., FREED, D., AND SCHWARTZ, A. (1973) J. Biol. Chem. 248, 7762-7772. 20. MERMIER, P., AND HASSELBACH, W. (1976) Eur. J. Biochem. 64, 613-620.
