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Partial purification and reconstitution of the (Ca2+ + Mg2+)-ATPase of erythrocyte membranes
hCHIVE.¶
OF
BIOCHEMISTRY AND BIOPHYSICS
Vol. 186, No. 1, February, pp. 202-210, 1978
Partial Purification
and Reconstitution of the (Ca2+ + Mg2+)-ATPase Erythrocyte Membranes
of
SCOTT W. PETERSON, PETER RONNER, AND ERNEST0 CARAFOLI Laboratory of Biochemistry, Swiss Federal Znstitute of Technology (ETH), Ztirich, Switzerland Received July 25, 1977; revised November 9, 1977 The (CaZ+ + MgZ+)-ATPase from human erythrooyte plasma membranes was partially purified and reconstituted by two different methods. Those proteins which were solubilixed from the membrane by Triton X-100 either were selectively incorporated into liposomes with a defined lipid content or were isoelectrically focused prior to reincorporation. In both types of reconstitution, maximum enzyme reactivation occurred when the vesicles consisted of acidic lipids, especially phosphatidyl serine. In the isoelectrically focused system, optimal enzyme activity was found to occur when the liposomes contained approximately 90 ~01 of phosphatidyl serine/mg of protein. By comparing enzyme activity in the isoelectrically focused, reconstituted system with that for a control detergent-protein system, it is estimated that the Ca*+-ATPaae was puriiied approximately &-fold.
The successful physiological functioning of the human erythrocyte depends in part on its ability to generate a low intracellular calcium (Ca2+)ion concentration of lo-' M (1, 2). Maintenance of this Ca2+ level is partly due to the low passive Ca2+ permeability of this system (31, but it is also caused by an active Ca2+transport system, which is present in the membrane of red blood cells (3, 4). Erythrocyte plasma membranes contain a (Ca2+ + Mg2+)-dependent ATPase (5, 6) and evidence suggests that this enzyme is the transport protein itself (2, 7). This Ca2+-ATPase composes about 0.02% of the total membrane protein and has a probable molecular weight of about 130,000 (8). To this date, the study of this physiologically important enzyme has proceeded along two lines: enzyme purification and enzyme lipid requirement. Some attempts to purify the enzyme in an active state have been reported (9). However, it has been found in this laboratory that the procedures already in print yield preparations which are contaminated by membrane proteins not considered to be associated with Ca2+-ATPase activity, such as band 3. Other researchers have investigated the
phospholipid requirement of the enzyme by the action of different phospholipases on the intact plasma membrane (10, 11). These reports conflict as to exactly which lipids are essential for this enzyme’s activity. Reconstitution of even a partially purified Ca2+-ATPase in vesicular phospholipid bilayers, which would further aid in understanding the relationship between this enzyme and its membrane environment, has not yet been reported. We present here the results of reconstitution attempts of an erythrocyte plasma membrane (Ca2+ + Mg2+)-ATPase which has been partially purified in different ways. The reconstituted vesicles have been characterized with regard to the effects of their lipid compositions on enzyme activity. Preliminary attempts to purify the enzyme protein by isoelectric focusing in a nonionic detergent suggest that this may be a successful general alternative method to membrane protein isolation. MATERIALS
202 0003-9861/78/1861-0202$,02.00/O Copyright 0 1978 by Academic press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Bed blood cell plasma membranes were prepared according to the method of Banner et al. (10) with some modifications. Lysing procedures were repeated at least 10 times to obtain membranes that
RECONSTITUTION
OF THE ERYTHROCYTE
appeared white or slightly pink. The final sediments were resuspended in 130 mu KC1 and 20 ml of Tris’ (pH 7.4) and stored at -80°C until used. For protein purification and reconstitution studies, these membranes (4.6 mg of protein/ml) were solubilixed with a solution which contained 0.3% Triton X-100 (1.5 mg of protein/mg of Triton X-1001 (Merck), 1 nne Tris (pH 7.4), 25 nne KCl, 1 ~llw MgCl,, and 5 PM CaCl, at 0°C for 10 min and were then centrifuged at 100,OOOgfor 1 h at 0°C. The supernatant was collected for the succeeding experiments. Reconstitution was done under the following conditions. The various lipid species used (Lipid Products) were dried to a solid phase under nitrogen and combined with protein solutions, either directly from the solubilised membrane supematant or from the isoelectric focusing fractions examined. After each solution was sonicated for 1 min at 0°C it was neutralized to pH 7.4. Volumes of 1 to 2 ml of the lipid-protein-detergent solutions were placed on a lO-ml Bio-Bead (Bio-Rad) column which had been equilibrated with 10 mM Tris (pH 7.4), 65 mM KCl, 1 rnru MgCl*, and 5 pu CaCl,. Fractions (1 ml) of the Bio-Bead eluant were then either analyzed for Ca2+-ATPase activity and protein and phospholipid content or concentrated and added to a Sepharose 4B column which had been equilibrated with the same medium that was used in the Bio-Bead column. Fractions of 1 ml from the Sepharose column were then analyzed in the same manner as the fractions from the Bio-Bead column. Isoelectric focusing was done in 2.5ml focusing columns on a 0.43-2% Ampholine gradient and a 520% sucrose gradient according to the method of Behnke et al. (12) with some modifications. The solubilixed membrane supematant was first concentrated in an Amicon ultrafiltration cell to 10 times its initial value, and 0.5 ml of this concentrated supematant was added to 2.5 ml (total volume) of focusing components. Therefore, in addition to the focusing components, the focusing solution consisted of 1 mg of protein/ml, 0.5% Triton X-100, 0.4 nne Tris (pH 7.41, 2.5 mu KCl, 0.2 mM MgCl,, and 0.1 FM CaCl,. Isoelectric focusing was done at 4°C for 16 h at a constant 200 V, and fractions of the focused column were collected. Enzyme assays were performed both before and after the reconstitution procedure. ATPase activity was estimated using a coupled enzyme assay according to the method of Ronner et al. (101in a medium containing 40 II~M Hepes buffer (pH 7.4). Phospho1 Abbreviations used: Tris, tris(hydroxymethyllaminomethane; Hepes, N-2-hydroxyethylpiperazinc-N’-2-ethanesulfonic acid; PS, phosphatidyl serine; PC, phosphatidyl choline; PE, phosphatidyl ethanolamine; OA, oleic acid; PEP, phosphoenolpyruvate; SDS, sodium dodecyl sulfate; EGTA, [ethylenebis(oxoethylenenitrilo)]tetraacetic acid.
Ca*+-ATPase
203
lipids were present in the amounts found in the various protein solutions. Before protein and phospholipid assays and gel electrophoresis, the fractions of the isoelectric focusing column were dialyzed against two changes of distilled water for 48 h to remove Ampholine. Protein assays were done according to the method of Lowry et al. (131, and phosphate assays were done according to the method of Lowry et al. (141. Gel electrophoresis was performed using a 4% focusing gel and an 8% separation gel in a 0.1% SDS solution according to the method of Meissner et al. (15). Spectrophotometric gel scanning was done at 280 nm. RESULTS
Partial protein purifiation by differential membrane protein solubilization and selective protein incorporation into liposomes. Under the conditions used, Triton X-100 solubilized mainly bands 3, 4.5, and 7 [protein bands are numbered according to Steck (IS)] and almost all of the accountable ATPase activity (Table I). The superTABLE I ENZYME ACTIVITIES DIJRING PARTIAL PURIFICATION AND RECONSTITUTION OF THE ERYTHIUXYTE PLASMA MEMBRANE Ca2+-ATPasea
Sample
Specific activity (.w$
Plasma membrane Pellet* SupernatantC 1 h after solubilization 3 h after solubilisation +Triton X-100 -Triton X-100d +ps +PE +pc +OA
0.66 0.10
8.52 0.78
100 9
1.8
5.0
62
0.60 0.10 2.25 0.00 0.10 0.90
1.66 0.28 1.96 0.00 0.17 1.19
20 3 23 0 2 14
Total activity (Pgol/
Percentage of total original activity
a The purification procedure consisted of differential protein eolubilization from the membrane and selective protein incorporation into liposomes. * The pellet was formed by centrifugation of erythrocyte plasma membranes that were solubilised with 0.3% Triton X-100. ’ The supematant was formed from the centrifugation described in Footnote 5. d Triton X-100 was removed and the vesicles were reconstituted according to the procedure described under Materials and Methods.
204
PETERSON.
RONNER.
natant was combined with varying amounts of lipid, briefly son&ted and neutralized to pH 7.4, and then placed on a lo-ml Bio-Bead column to remove Triton X-100 and eluted with buffer. The active fractions, suitably concentrated, were placed on a Sepharose 4B column to separate liposomes from free proteins. Figure 1 shows a representative electron micrograph of the Sepharose eluant when the Triton removal was done at 4°C. All eluants showed the presence of large, monolamellar vesicles varying in size from 500 A to 0.1 pm in diameter. The presence of these vesicles demonstrates that the reduction in Triton X-100 concentration to O.Ol%, as achieved on the Bio-Bead column (171, is sufficient to allow the generation of lipid bilayer vesicles. By performing the Triton removal at 23”C, the vesicles became more consistent in size, mostly in the range of 2000 to 4000 A in diameter. Figure 2 shows a typical SDS gel scan from which the histogram shown in Fig. 3 was derived. As was mentioned earlier, this histogram shows that solubilization of the plasma membrane caused significant amounts of protein bands 3, 4.5, and 7 (and hemoglobin) and 90% of the accountable (Ca2+ + Mg2+)-ATPase activity to be released into the supernatant (Table I). When these solubilized proteins were reincorporated into liposomes and separated from unincorporated proteins on a Sepharose 4B column, there was further selection of bands 5, 6, and 7. There was a decrease in the contribution of protein in the liposomes by band 4.5, although this band still contributed 13% of the total membrane protein. Protein incorporation into liposomes, when done at 23”C, showed no significant changes in distribution pat-
AND
CARAFOLI
+ Me+)-ATPase enzyme increased 3.5 times over that of the original plasma membrane. The observation that enzyme activity decreased when the lipid to protein ratio increased past the optimal level (Fig. 4) is not easily explained. The gel protein patterns show that the liposomes in each of the fractions of the Sepharose column all had the same relative protein distributions. Although this is only indirect evidence that the (Ca2+ + Mg2+)-ATPase would also be found in the same relative proportions, it seems that a decrease in (Ca2+ + Mg2+)-ATPase in the latter fractions is not the likely reason for the observation. This relationship between lipid and enzyme is further supported by the fact that, when the experiments were done at varying beginning ratios of lipid to protein (and, hence, different ratios in the eluting fractions), variations in maximal activity were seen (Table II). The greatest maximal activity was seen at a final ratio of 18 -01 of lipid/mg of protein. In theory, the observed inactivity in the phosphatidyl ethanolamine liposomes may be a result of an unfavorable asymmetric distribution of the enzyme. Therefore, these liposomes were resolubilized with Triton X-100, and enzyme activities were measured (Fig. 5). Should the protein be oriented in the bialyer so that it is unable to hydrolyze the ATP in the medium, then this resolubilization of the liposomes would release the enzyme from the membrane constraints and enzyme activity would be recorded. Although the results from some of the inactive systems, such as the phosphatidyl choline liposomes, could be interpreted using the above reasoning, other systems such as the phosphatidyl serine and oleic acid liposomes tt?llW. The reconstituted system had (Ca2+ + do not lend themselves easily to this explanation. The reasons behind the observed Mg2+)-ATPase, and it was observed that the (Ca2+ + Mg2+)-ATPase activity varied Triton resolubilization effects may thus be with the phospholipid species present and more complex, involving detergent-prowith changes in the ratio of lipid to pro- tein-lipid micellar environments. To determine the effect of reconstitution tein. For the system which had the maxitemperature on the enzyme activities, remal activity (phosphatidyl serine), the ratio of 20 pm01 of phospholipid/mg of pro- constitution of phosphatidyl serine lipotein yielded optimal activity (Fig. 4). At somes was performed at 23°C (Figs. 4 and this ratio, the specific activity for the (Ca2+ 6). The maximal specific activity was 50%
RECONSTITUTION
OF THE
ERYTHROCYTE
Ca*+-ATPase
205
206
PETERSON, RONNER, AND CARAFOLI
0
0
i C
0
J 40 MIGRATION
I
I
so
120
I
150
(mm)
FIG. 2. Erythrocyte plasma membrane proteins. Shown is a spectrophotometric scan of an SDS gel stained for protein with Coomassie blue aRer electrophoresis of 50 pg of erythrocyte plasma membrane protein. The procedures are described in Ref. 15. Protein bands are labeled according to Steck (16).
PROTEIN
BAND
FIG. 3. Relative protein species distribution during partial puritication of (Ca*+ + MgP+)ATPase. Puritication was by differential protein solubilization and selective protein incorporation into liposomes. Reconstitution was done at 0°C. The membrane protein species distribution for the erythrocyte plasma membrane 0, the Triton X-100~solubilized supernatant from the plasma membrane (a), and the phosphatidyl serine-reconstituted system W are shown. Gels were scanned spectrophotometrically at 280 nm and the areas under the resulting gel scans were integrated to derive these data. Protein bands are labeled according to Steck (16).
less when the reconstitution was done at 23°C than when it was done at 0°C. As mentioned before, the protein incorporation patterns of the two systems were the same. Partial purification by differential membrane protein solubilization, isoelectric focusing, and selective protein incorporation into liposomes. The Triton X-100 solution containing the proteins which had
been selectively solubilized from the plasma membranes was isoelectrically focused for 16 h. The solution in this column showed almost no (Ca2+ + Mg3+)-ATPase activity after the focusing (Table III). On the basis of the results in the previous section, reconstitutions of fractions of the isoelectric focusing column were done with various phospholipids at 0°C. Electron microscopy of the phospholipid-reconsti-
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OF THE ERYTHROCYTE
FIG. 4. The relationship between (Ca*+ + Mg*+)ATPase activity and the phosphatidyl serine to protein ratio. The enzyme was partially purified by differential membrane protein solubilization and selective protein incorporation into liposomes. Reconstitution was done at 0°C. ATPase activity was measured at 340 nm using a coupled enzyme assay in a medium containing about 150 pg of membrane protein, 60 mM Hepes (pH 7.4), 240 mM KCl, 5 PM CaCl,, 10 mr.r MgCh, 0.4 mM NADH, 1.0 mM PEP, lactate dehydrogenase (1 III), and pyruvate kinase (1 IU) in a total volume of 1 ml at 37°C. ARer this initial slope was determined, EGTA (1 mM) was added and the (Ca*+ + MgZ+)-ATPase activity was calculated from the difference of the two slopes.
Ca2+-ATPase
207
The specific activity for the (Ca2+ + Mg2+)ATPase enzyme of the system reconstituted with phosphatidyl serine was increased 14 times over that of the original plasma membrane. Examinations by gel electrophoresis of protein species that are present in Sepharose fractions (Fig. 8) showed that isoelectric focusing and differential incorporation into liposomes led to a selective enhancement of protein bands 6 and 7 in the isoelectric focusing fraction that contained (Ca2+ + Mg2+)-ATPase activity. In the same fraction there was a decrease in the contribution of bands 3 and 4.2 to the total protein content.
TABLE II EFFECT OF VARIATIONS IN THE INITIAL PHOSPHATIDYL SERINE TO PROTEIN RATIO ON OPTIMAL Ca2+-ATPase ACTIVITY
PS (pmol)a protein (mg) 0.88
Maximum specific activity (pmol/mg/h) 0.00
PS (rmol)* protein (mg) 0.00
6.2 18.3 38.8
0.00 0.51 2.22
18.9
61.6
1.00
28.0
0.00 8.9
a Ratio of PS to protein before addition to the BioBead column. b Ratio of PS to protein in the Sepharose fraction with maximum specific activity.
tuted, Sepharose-excluded fractions showed liposomes with the same characteristics as those seen before. Using phosphatidyl serine or oleic acid for reconstitution (Table III), enzymatitally active fractions could be localized between pH 4 and 7 of the isoelectric focusing column. The ratio of lipid to protein was investigated in the phosphatidyl serine system, and the maximal specific activity was found at a ratio of 90 pmol of phosphatidyl serine/mg of protein (Fig. 7).
LIPID-ENZYME
SYSTEM
FIG. 5. Effect of Triton X-100 resolubilization
of reconstituted systems on erythrocyte plasma membrane (Ca*+ + Mgl+)-ATPase enzyme activity. Reconstituted systems were resolubilized with 0.5% Triton X-100 (m) and the activities were compared with those for the unsolubilized lipid-enzyme systems (0). Also included are enzyme activities for the (Caz+ + Mg*+)-ATPase in the original plasma membrane (PM) and in the Triton-solubilized supernatant (SN). ATPase activities were measured by the procedure described in Fig. 4.
FIG. 6. The relationship between (CaZ+ + M&+)ATPase activity and the phosphatidyl serine to protein ratio. The enzyme was partially purified by the same procedure described in Fig. 3. Reconstitution was done at 23°C. ATPase activity was measured by the procedure described in Fig. 4.
208
PETERSON, RONNER, AND CARAFOLI TABLE III
Me+)-ATPase enzyme activity was found to occur in vesicular membrane systems reconstituted both from Triton X-lOO-solubilized erythrocyte plasma membrane proSample Specitic Total Percentteins and from a preparation partially puractivity activity age of ified by isoelectric focusing. Larger liposototal km011 (pn$/ ma1 particles, more regular in size and original m&h) activity still monolamellar, could be made by removing the detergent, and thereby reconF’;ey; membrane 0.66 0.10 8.62 0.78 1009 stituting the system, at room temperature. Supematant* 1 h after solubiliza1.8 However, this results in lower specific 5.0 62 tion activity, likely due to partial inactivation 20 h after solubilizaof the ATPase. This rationale is reinforced tion +Triton X-100 0.14 0.85 10 by the observation that no significant -Triton X-100” changes in protein distribution patterns occur when the temperature of reconstitu+PS +PE 8.94 0.00 0.77 0.00 i +PC 0.00 0.00 0 tion is raised. It should be noted that +OA 3.82 0.08 1 bilayer vesicles could be formed from oleic D The purification procedure consisted of differacid dispersions, an observation first reential membrane protein solubilization, isoelectric ported by i3ebicki and Hicks (18). focusing, and selective protein incorporation into The reliability of the reconstitution liposomes. technique is demonstrated by the similar* The pellet and supematant were from the cenity of the lipid effects on enzyme activity trifugation described in Table I, Footnote b. between reconstituted lipid-protein sysc Triton X-100 was removed and the vesicles were tems that have and have not been isoereconstituted according to the procedure described lectrically focused. However, even though under Materials and Methods. one would expect that the amount of lipid required to generate a lipid environment for a particular protein would decrease as the amount of extraneous membrane protein is removed from the system, the opposite effect was observed. Ronner et al. (10) and Roelofsen and Schatzmann (11) both studied the lipid requirement of this enzyme by using phospholipase, but they obtained discrepant results. In particular, Roelofsen and FIG. 7. The relationship between (Ca2+ + M&+1Schatzmann found a reactivation with all ATPase activity and the phosphatidyl serine to phospholipids studied in the present paprotein’ratio. The enzyme was partially purified by per, but not with free fatty acids such as differential protein solubilization, isoelectric focus- oleic acid. The experiments reported here, ing, and selective protein incorporation into lipowhich have covered the same lipid-requirsomes. Reconstitution was done at 0°C. ATPase ing parameters by reconstitution techactivity was measured by the procedure described niques, have supported the work of Ronner in Fig. 4. et al. (10). Reactivation of enzyme activity is more pronounced when the lipid phosDISCUSSION phatidyl serine or the fatty acid is present. The effective ability to judge whether or The information in this paper deals with not a membrane enzyme has been purified the reactivation of the human erythrocyte (Ca2+ + MgZ+)-ATPase enzyme by success- typically rests on two criteria: the ability fully reconstituting this protein in a par- to visualize, by gel electrophoresis, an tially purified form in a membrane system increase in the amount of enzyme after with a defined lipid medium. (Caz+ + the isolation procedure, and the increase ENZYME ACTIVITIES DIJBING PARTIAL PIJRIFICATION AND RECONSTITUTION OF THE ERYTHROCYTE ~BMA MEMBRANE Ca2+-ATPase’
RECONSTITUTION
OF THE ERYTHROCYTE
Ca*+-ATPase
209
FIG. 8. Relative protein species distribution during partial purification of the (Ca2+ + Mg2+)-ATPase. Purification was done under the same conditions described in Fig. 7. The membrane protein species distribution for the erythrocyte plasma membrane 0, the Triton X-100.solubilized supematant from the plasma membranes (as), and the phosphatidyl aerinereconstituted system after isoelectric focusing (a) are shown. Analysis of data was by the same procedure described in Fig. 3.
in specific activity of the solution as the enzyme is purified. The first method of determination is difficult to use in this situation because this protein comprises only about 0.02% of the total membrane protein (8). A purification of 500-fold would probably be necessary to visualize this enzyme effectively, and such a purification has yet to be achieved. The second criterion is ambiguous for this particular enzyme. Our results have shown that this (Ca2+ + Me+)-ATPa.se has activities that vary significantly, depending on the amount and type of lipid present. Furthermore, unreported observations in our laboratory have shown that the (Ca2+ + Mg2+)-ATPase is extremely labile with time in Triton X-100. An increase (or even a decrease) in specific activity during a purification step may therefore be a product of three different factors: protein purification, enzyme activation due to the presence of a necessary cofactor, and enzyme inactivation because of protein lability. Therefore, to judge the success of the attempts at purification of this protein, we have chosen to correlate the presence of enzyme activity, regardless of its quantity, with the selective presence of the more readily recognizable protein species of the erythrocyte plasma membrane. This (Ca2+ + Mg2+)-ATPase has been reported by Knauf et al. (8) to have a molecular weight of approximately 150,000, and it should be stressed that an observation of the enrichment in bands of any other molecular weight is not necessary to be construed as a statement of association between the ATPase and that band. In this paper, we have demonstrated
several methods of partially purifying a membrane enzyme. The partial purikation of erythrocyte plasma membrane proteins by selective solubilization with detergent has already been reported by Wolf and Gietzen (19). Selective protein incorporation into liposomes occurs when the supernatant has the detergent removed in the presence of phosphatidyl serine, and the resulting liposomes are separated from free proteins. Isoelectric focusing separates proteins on the basis of their isoelectric points (20). If membrane proteins which have been solubilized in a nonionic detergent such as Triton X-100 are used, these proteins can be separated on the basis of charges that they show while in the micelle. The most noticeable contribution of isoelectric focusing to protein was the decrease of band 3 in the fraction which contained (Ca2+ + Mg2+)-ATPase activity. The dominant protein species after this series of partial purifkations are bands 4.5, 6, and 7. It must be noted that the total (Ca2+ + Mg2+)-ATPase activity in this optimal system is only 9% of that found in the supernatant of the Triton-solubilized plasma membranes (Table III). The enzyme has been found to be inactivated by this detergent with time, and it is assumed that this is the reason for the low yield. In fact, 89% of the total activity in the original supernatant, maintained at 4°C for 24 h in 0.3% Triton X100, is found after isoelectric focusing. This suggests that almost all of the activity of the enzyme in the debilitating environment of the detergent has been accounted for. It is possible, therefore, that much of the go-fold increase in specific
210
PETERSON, RONNER, AND CARAFOLI
activity over this supernatant control by the phosphatidyl serine liposomal system represents a true purification. Because of the properties of the acidic lipid present, it is di&ult to measure Ca2+ transport in the enzymatically active reconstituted systems. These lipids bind calcium ions, which in turn induce transition temperature-like phase changes in the lipid hydrocarbon region and vesicle fusion (21). Work is presently under way to develop a suitable lipid mixture that will promote both enzyme activity and Ca2+ transport. The isoelectric focusing procedure is not yet refined, as witnessed by the large pH range that must be collected to reconstitute an active membrane enzyme. The results reported here were done on small focusing columns, which were more advantageous than a larger column with respect to the amount of focusing components required and the running times of the systems. The major limitation of such a system is that only small amounts of membrane material may be used, thus making the degree of resolution of the experiments less precise. Work is presently under way to adapt the procedures that were used in these small columns to larger systems. ACKNOWLEDGMENTS The authors would like to thank Dr. M. Miiller, Service for Electron Microscopy, ETH Zurich, for preparing the electron microscopy pictures and the Blutspendezentrum Limmattal for supplying the human red blood cells. This project was funded by a Swiss National Science Foundation grant (No. 3.592-0.75) and by a Forschungsprojekt, ETH (No. 0.330.076.60/5). REFERENCES 1. WEED, R. I., AND CHAILLEY, B. (1973) in Red
Cell Shape (Bessis, M., Weed, R. I., and
Leblond, P. F., eds.), pp. 55-68, SpringerVerlag, Berlin/New York. 2. SCHATZMANN,H. J. (1975) in Current Topics in Membranes and Transport (Banner, F., and Kleinzeller, A., eds.), Vol. 6, pp. 125-168, Academic Press, New York. 3. S%XIATZMANN, H. J., AND VINCENZI, F. F. (1969). J. Physiol. (London) 201, 369-395. 4. SCHATZMANN,H. J. (1966) Experientia 22, 364368. 5. WINS, T., AND S~HOFFENIEL~, E. (1966)B&him. Biophys. Actu 120, 341-350. 6. SCHATZMANN, H. J., AND ROSSI, G. L. (1971) B&him. Biophys. Acta 241, 379-392. 7. WOLF, H. U. (1972)B&him. Biophys. Actu 266, 361. 8. KNAUF, P. A., Paovsamo, F., AND HOFFMAN,J. F. (1974) J. Gen. Physiol. 63, 324-336. 9. WOLF, H. U., Dnvxvoss, G., AND LICHTNEB, R. (1976)Experientia 32, 776. 10. R~NNEB, P., GAZZO~I, P., AND CARAFOLI, E. (1977) Arch. Biochem. Biophys. 179, 578-583. 11. ROEMFBEN, B., AND SCHATZMANN, H. J. (1977) B&him. Biophys. Actu 464, 17-36. 12. BEHNKE, J. N., DAGHER, S. M., MAEBEY, T. H., AND DEAL, W. C. (1975) Anal. Biochem. 69, l-9. 13. LOWRY, 0. H., ROSEBIKBUGH,N. J., FAIR, A. L., AND RANDALL, R. J. (1951) J. Bid Chem. 193, 265-275. 14. LOWRY, 0. H., ROBERTS, N. R., LEINER, K. Y., WV, M., AND FARR, A. L. (1954) J. Biol. Chem. 207, 1-18. 15. MEI~~NER, G., CONNER, G. E., AND FLEIBCHER, S. (1973) B&him. Biophys. Actu 298, 246269. 16. STECK,T. L. (1974) J. Cell Bid. 62, l-19. 17. HOLLOWAY, P. W. (1973) And. Biochem. 53, 304-308. 18. GEBICKI, J. M., AND Hnxs, M. (1976) Chcm. Phys. Lipids 16, 142-160. 19. WOLF, H. U., AND GIETZEN, K. Z. (1974)PhysioZ. Chem. 356, 1272. 20. VESTJCBBEBG, 0. (1971) Methods Enzymol. 22, 389-412. 21. HAUSER, H., FINER, E. G., AND DARKE, A. (1977) Biochem. Biophys. Res. Commun. 76,267-274.
OF
BIOCHEMISTRY AND BIOPHYSICS
Vol. 186, No. 1, February, pp. 202-210, 1978
Partial Purification
and Reconstitution of the (Ca2+ + Mg2+)-ATPase Erythrocyte Membranes
of
SCOTT W. PETERSON, PETER RONNER, AND ERNEST0 CARAFOLI Laboratory of Biochemistry, Swiss Federal Znstitute of Technology (ETH), Ztirich, Switzerland Received July 25, 1977; revised November 9, 1977 The (CaZ+ + MgZ+)-ATPase from human erythrooyte plasma membranes was partially purified and reconstituted by two different methods. Those proteins which were solubilixed from the membrane by Triton X-100 either were selectively incorporated into liposomes with a defined lipid content or were isoelectrically focused prior to reincorporation. In both types of reconstitution, maximum enzyme reactivation occurred when the vesicles consisted of acidic lipids, especially phosphatidyl serine. In the isoelectrically focused system, optimal enzyme activity was found to occur when the liposomes contained approximately 90 ~01 of phosphatidyl serine/mg of protein. By comparing enzyme activity in the isoelectrically focused, reconstituted system with that for a control detergent-protein system, it is estimated that the Ca*+-ATPaae was puriiied approximately &-fold.
The successful physiological functioning of the human erythrocyte depends in part on its ability to generate a low intracellular calcium (Ca2+)ion concentration of lo-' M (1, 2). Maintenance of this Ca2+ level is partly due to the low passive Ca2+ permeability of this system (31, but it is also caused by an active Ca2+transport system, which is present in the membrane of red blood cells (3, 4). Erythrocyte plasma membranes contain a (Ca2+ + Mg2+)-dependent ATPase (5, 6) and evidence suggests that this enzyme is the transport protein itself (2, 7). This Ca2+-ATPase composes about 0.02% of the total membrane protein and has a probable molecular weight of about 130,000 (8). To this date, the study of this physiologically important enzyme has proceeded along two lines: enzyme purification and enzyme lipid requirement. Some attempts to purify the enzyme in an active state have been reported (9). However, it has been found in this laboratory that the procedures already in print yield preparations which are contaminated by membrane proteins not considered to be associated with Ca2+-ATPase activity, such as band 3. Other researchers have investigated the
phospholipid requirement of the enzyme by the action of different phospholipases on the intact plasma membrane (10, 11). These reports conflict as to exactly which lipids are essential for this enzyme’s activity. Reconstitution of even a partially purified Ca2+-ATPase in vesicular phospholipid bilayers, which would further aid in understanding the relationship between this enzyme and its membrane environment, has not yet been reported. We present here the results of reconstitution attempts of an erythrocyte plasma membrane (Ca2+ + Mg2+)-ATPase which has been partially purified in different ways. The reconstituted vesicles have been characterized with regard to the effects of their lipid compositions on enzyme activity. Preliminary attempts to purify the enzyme protein by isoelectric focusing in a nonionic detergent suggest that this may be a successful general alternative method to membrane protein isolation. MATERIALS
202 0003-9861/78/1861-0202$,02.00/O Copyright 0 1978 by Academic press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Bed blood cell plasma membranes were prepared according to the method of Banner et al. (10) with some modifications. Lysing procedures were repeated at least 10 times to obtain membranes that
RECONSTITUTION
OF THE ERYTHROCYTE
appeared white or slightly pink. The final sediments were resuspended in 130 mu KC1 and 20 ml of Tris’ (pH 7.4) and stored at -80°C until used. For protein purification and reconstitution studies, these membranes (4.6 mg of protein/ml) were solubilixed with a solution which contained 0.3% Triton X-100 (1.5 mg of protein/mg of Triton X-1001 (Merck), 1 nne Tris (pH 7.4), 25 nne KCl, 1 ~llw MgCl,, and 5 PM CaCl, at 0°C for 10 min and were then centrifuged at 100,OOOgfor 1 h at 0°C. The supernatant was collected for the succeeding experiments. Reconstitution was done under the following conditions. The various lipid species used (Lipid Products) were dried to a solid phase under nitrogen and combined with protein solutions, either directly from the solubilised membrane supematant or from the isoelectric focusing fractions examined. After each solution was sonicated for 1 min at 0°C it was neutralized to pH 7.4. Volumes of 1 to 2 ml of the lipid-protein-detergent solutions were placed on a lO-ml Bio-Bead (Bio-Rad) column which had been equilibrated with 10 mM Tris (pH 7.4), 65 mM KCl, 1 rnru MgCl*, and 5 pu CaCl,. Fractions (1 ml) of the Bio-Bead eluant were then either analyzed for Ca2+-ATPase activity and protein and phospholipid content or concentrated and added to a Sepharose 4B column which had been equilibrated with the same medium that was used in the Bio-Bead column. Fractions of 1 ml from the Sepharose column were then analyzed in the same manner as the fractions from the Bio-Bead column. Isoelectric focusing was done in 2.5ml focusing columns on a 0.43-2% Ampholine gradient and a 520% sucrose gradient according to the method of Behnke et al. (12) with some modifications. The solubilixed membrane supematant was first concentrated in an Amicon ultrafiltration cell to 10 times its initial value, and 0.5 ml of this concentrated supematant was added to 2.5 ml (total volume) of focusing components. Therefore, in addition to the focusing components, the focusing solution consisted of 1 mg of protein/ml, 0.5% Triton X-100, 0.4 nne Tris (pH 7.41, 2.5 mu KCl, 0.2 mM MgCl,, and 0.1 FM CaCl,. Isoelectric focusing was done at 4°C for 16 h at a constant 200 V, and fractions of the focused column were collected. Enzyme assays were performed both before and after the reconstitution procedure. ATPase activity was estimated using a coupled enzyme assay according to the method of Ronner et al. (101in a medium containing 40 II~M Hepes buffer (pH 7.4). Phospho1 Abbreviations used: Tris, tris(hydroxymethyllaminomethane; Hepes, N-2-hydroxyethylpiperazinc-N’-2-ethanesulfonic acid; PS, phosphatidyl serine; PC, phosphatidyl choline; PE, phosphatidyl ethanolamine; OA, oleic acid; PEP, phosphoenolpyruvate; SDS, sodium dodecyl sulfate; EGTA, [ethylenebis(oxoethylenenitrilo)]tetraacetic acid.
Ca*+-ATPase
203
lipids were present in the amounts found in the various protein solutions. Before protein and phospholipid assays and gel electrophoresis, the fractions of the isoelectric focusing column were dialyzed against two changes of distilled water for 48 h to remove Ampholine. Protein assays were done according to the method of Lowry et al. (131, and phosphate assays were done according to the method of Lowry et al. (141. Gel electrophoresis was performed using a 4% focusing gel and an 8% separation gel in a 0.1% SDS solution according to the method of Meissner et al. (15). Spectrophotometric gel scanning was done at 280 nm. RESULTS
Partial protein purifiation by differential membrane protein solubilization and selective protein incorporation into liposomes. Under the conditions used, Triton X-100 solubilized mainly bands 3, 4.5, and 7 [protein bands are numbered according to Steck (IS)] and almost all of the accountable ATPase activity (Table I). The superTABLE I ENZYME ACTIVITIES DIJRING PARTIAL PURIFICATION AND RECONSTITUTION OF THE ERYTHIUXYTE PLASMA MEMBRANE Ca2+-ATPasea
Sample
Specific activity (.w$
Plasma membrane Pellet* SupernatantC 1 h after solubilization 3 h after solubilisation +Triton X-100 -Triton X-100d +ps +PE +pc +OA
0.66 0.10
8.52 0.78
100 9
1.8
5.0
62
0.60 0.10 2.25 0.00 0.10 0.90
1.66 0.28 1.96 0.00 0.17 1.19
20 3 23 0 2 14
Total activity (Pgol/
Percentage of total original activity
a The purification procedure consisted of differential protein eolubilization from the membrane and selective protein incorporation into liposomes. * The pellet was formed by centrifugation of erythrocyte plasma membranes that were solubilised with 0.3% Triton X-100. ’ The supematant was formed from the centrifugation described in Footnote 5. d Triton X-100 was removed and the vesicles were reconstituted according to the procedure described under Materials and Methods.
204
PETERSON.
RONNER.
natant was combined with varying amounts of lipid, briefly son&ted and neutralized to pH 7.4, and then placed on a lo-ml Bio-Bead column to remove Triton X-100 and eluted with buffer. The active fractions, suitably concentrated, were placed on a Sepharose 4B column to separate liposomes from free proteins. Figure 1 shows a representative electron micrograph of the Sepharose eluant when the Triton removal was done at 4°C. All eluants showed the presence of large, monolamellar vesicles varying in size from 500 A to 0.1 pm in diameter. The presence of these vesicles demonstrates that the reduction in Triton X-100 concentration to O.Ol%, as achieved on the Bio-Bead column (171, is sufficient to allow the generation of lipid bilayer vesicles. By performing the Triton removal at 23”C, the vesicles became more consistent in size, mostly in the range of 2000 to 4000 A in diameter. Figure 2 shows a typical SDS gel scan from which the histogram shown in Fig. 3 was derived. As was mentioned earlier, this histogram shows that solubilization of the plasma membrane caused significant amounts of protein bands 3, 4.5, and 7 (and hemoglobin) and 90% of the accountable (Ca2+ + Mg2+)-ATPase activity to be released into the supernatant (Table I). When these solubilized proteins were reincorporated into liposomes and separated from unincorporated proteins on a Sepharose 4B column, there was further selection of bands 5, 6, and 7. There was a decrease in the contribution of protein in the liposomes by band 4.5, although this band still contributed 13% of the total membrane protein. Protein incorporation into liposomes, when done at 23”C, showed no significant changes in distribution pat-
AND
CARAFOLI
+ Me+)-ATPase enzyme increased 3.5 times over that of the original plasma membrane. The observation that enzyme activity decreased when the lipid to protein ratio increased past the optimal level (Fig. 4) is not easily explained. The gel protein patterns show that the liposomes in each of the fractions of the Sepharose column all had the same relative protein distributions. Although this is only indirect evidence that the (Ca2+ + Mg2+)-ATPase would also be found in the same relative proportions, it seems that a decrease in (Ca2+ + Mg2+)-ATPase in the latter fractions is not the likely reason for the observation. This relationship between lipid and enzyme is further supported by the fact that, when the experiments were done at varying beginning ratios of lipid to protein (and, hence, different ratios in the eluting fractions), variations in maximal activity were seen (Table II). The greatest maximal activity was seen at a final ratio of 18 -01 of lipid/mg of protein. In theory, the observed inactivity in the phosphatidyl ethanolamine liposomes may be a result of an unfavorable asymmetric distribution of the enzyme. Therefore, these liposomes were resolubilized with Triton X-100, and enzyme activities were measured (Fig. 5). Should the protein be oriented in the bialyer so that it is unable to hydrolyze the ATP in the medium, then this resolubilization of the liposomes would release the enzyme from the membrane constraints and enzyme activity would be recorded. Although the results from some of the inactive systems, such as the phosphatidyl choline liposomes, could be interpreted using the above reasoning, other systems such as the phosphatidyl serine and oleic acid liposomes tt?llW. The reconstituted system had (Ca2+ + do not lend themselves easily to this explanation. The reasons behind the observed Mg2+)-ATPase, and it was observed that the (Ca2+ + Mg2+)-ATPase activity varied Triton resolubilization effects may thus be with the phospholipid species present and more complex, involving detergent-prowith changes in the ratio of lipid to pro- tein-lipid micellar environments. To determine the effect of reconstitution tein. For the system which had the maxitemperature on the enzyme activities, remal activity (phosphatidyl serine), the ratio of 20 pm01 of phospholipid/mg of pro- constitution of phosphatidyl serine lipotein yielded optimal activity (Fig. 4). At somes was performed at 23°C (Figs. 4 and this ratio, the specific activity for the (Ca2+ 6). The maximal specific activity was 50%
RECONSTITUTION
OF THE
ERYTHROCYTE
Ca*+-ATPase
205
206
PETERSON, RONNER, AND CARAFOLI
0
0
i C
0
J 40 MIGRATION
I
I
so
120
I
150
(mm)
FIG. 2. Erythrocyte plasma membrane proteins. Shown is a spectrophotometric scan of an SDS gel stained for protein with Coomassie blue aRer electrophoresis of 50 pg of erythrocyte plasma membrane protein. The procedures are described in Ref. 15. Protein bands are labeled according to Steck (16).
PROTEIN
BAND
FIG. 3. Relative protein species distribution during partial puritication of (Ca*+ + MgP+)ATPase. Puritication was by differential protein solubilization and selective protein incorporation into liposomes. Reconstitution was done at 0°C. The membrane protein species distribution for the erythrocyte plasma membrane 0, the Triton X-100~solubilized supernatant from the plasma membrane (a), and the phosphatidyl serine-reconstituted system W are shown. Gels were scanned spectrophotometrically at 280 nm and the areas under the resulting gel scans were integrated to derive these data. Protein bands are labeled according to Steck (16).
less when the reconstitution was done at 23°C than when it was done at 0°C. As mentioned before, the protein incorporation patterns of the two systems were the same. Partial purification by differential membrane protein solubilization, isoelectric focusing, and selective protein incorporation into liposomes. The Triton X-100 solution containing the proteins which had
been selectively solubilized from the plasma membranes was isoelectrically focused for 16 h. The solution in this column showed almost no (Ca2+ + Mg3+)-ATPase activity after the focusing (Table III). On the basis of the results in the previous section, reconstitutions of fractions of the isoelectric focusing column were done with various phospholipids at 0°C. Electron microscopy of the phospholipid-reconsti-
RECONSTITUTION
OF THE ERYTHROCYTE
FIG. 4. The relationship between (Ca*+ + Mg*+)ATPase activity and the phosphatidyl serine to protein ratio. The enzyme was partially purified by differential membrane protein solubilization and selective protein incorporation into liposomes. Reconstitution was done at 0°C. ATPase activity was measured at 340 nm using a coupled enzyme assay in a medium containing about 150 pg of membrane protein, 60 mM Hepes (pH 7.4), 240 mM KCl, 5 PM CaCl,, 10 mr.r MgCh, 0.4 mM NADH, 1.0 mM PEP, lactate dehydrogenase (1 III), and pyruvate kinase (1 IU) in a total volume of 1 ml at 37°C. ARer this initial slope was determined, EGTA (1 mM) was added and the (Ca*+ + MgZ+)-ATPase activity was calculated from the difference of the two slopes.
Ca2+-ATPase
207
The specific activity for the (Ca2+ + Mg2+)ATPase enzyme of the system reconstituted with phosphatidyl serine was increased 14 times over that of the original plasma membrane. Examinations by gel electrophoresis of protein species that are present in Sepharose fractions (Fig. 8) showed that isoelectric focusing and differential incorporation into liposomes led to a selective enhancement of protein bands 6 and 7 in the isoelectric focusing fraction that contained (Ca2+ + Mg2+)-ATPase activity. In the same fraction there was a decrease in the contribution of bands 3 and 4.2 to the total protein content.
TABLE II EFFECT OF VARIATIONS IN THE INITIAL PHOSPHATIDYL SERINE TO PROTEIN RATIO ON OPTIMAL Ca2+-ATPase ACTIVITY
PS (pmol)a protein (mg) 0.88
Maximum specific activity (pmol/mg/h) 0.00
PS (rmol)* protein (mg) 0.00
6.2 18.3 38.8
0.00 0.51 2.22
18.9
61.6
1.00
28.0
0.00 8.9
a Ratio of PS to protein before addition to the BioBead column. b Ratio of PS to protein in the Sepharose fraction with maximum specific activity.
tuted, Sepharose-excluded fractions showed liposomes with the same characteristics as those seen before. Using phosphatidyl serine or oleic acid for reconstitution (Table III), enzymatitally active fractions could be localized between pH 4 and 7 of the isoelectric focusing column. The ratio of lipid to protein was investigated in the phosphatidyl serine system, and the maximal specific activity was found at a ratio of 90 pmol of phosphatidyl serine/mg of protein (Fig. 7).
LIPID-ENZYME
SYSTEM
FIG. 5. Effect of Triton X-100 resolubilization
of reconstituted systems on erythrocyte plasma membrane (Ca*+ + Mgl+)-ATPase enzyme activity. Reconstituted systems were resolubilized with 0.5% Triton X-100 (m) and the activities were compared with those for the unsolubilized lipid-enzyme systems (0). Also included are enzyme activities for the (Caz+ + Mg*+)-ATPase in the original plasma membrane (PM) and in the Triton-solubilized supernatant (SN). ATPase activities were measured by the procedure described in Fig. 4.
FIG. 6. The relationship between (CaZ+ + M&+)ATPase activity and the phosphatidyl serine to protein ratio. The enzyme was partially purified by the same procedure described in Fig. 3. Reconstitution was done at 23°C. ATPase activity was measured by the procedure described in Fig. 4.
208
PETERSON, RONNER, AND CARAFOLI TABLE III
Me+)-ATPase enzyme activity was found to occur in vesicular membrane systems reconstituted both from Triton X-lOO-solubilized erythrocyte plasma membrane proSample Specitic Total Percentteins and from a preparation partially puractivity activity age of ified by isoelectric focusing. Larger liposototal km011 (pn$/ ma1 particles, more regular in size and original m&h) activity still monolamellar, could be made by removing the detergent, and thereby reconF’;ey; membrane 0.66 0.10 8.62 0.78 1009 stituting the system, at room temperature. Supematant* 1 h after solubiliza1.8 However, this results in lower specific 5.0 62 tion activity, likely due to partial inactivation 20 h after solubilizaof the ATPase. This rationale is reinforced tion +Triton X-100 0.14 0.85 10 by the observation that no significant -Triton X-100” changes in protein distribution patterns occur when the temperature of reconstitu+PS +PE 8.94 0.00 0.77 0.00 i +PC 0.00 0.00 0 tion is raised. It should be noted that +OA 3.82 0.08 1 bilayer vesicles could be formed from oleic D The purification procedure consisted of differacid dispersions, an observation first reential membrane protein solubilization, isoelectric ported by i3ebicki and Hicks (18). focusing, and selective protein incorporation into The reliability of the reconstitution liposomes. technique is demonstrated by the similar* The pellet and supematant were from the cenity of the lipid effects on enzyme activity trifugation described in Table I, Footnote b. between reconstituted lipid-protein sysc Triton X-100 was removed and the vesicles were tems that have and have not been isoereconstituted according to the procedure described lectrically focused. However, even though under Materials and Methods. one would expect that the amount of lipid required to generate a lipid environment for a particular protein would decrease as the amount of extraneous membrane protein is removed from the system, the opposite effect was observed. Ronner et al. (10) and Roelofsen and Schatzmann (11) both studied the lipid requirement of this enzyme by using phospholipase, but they obtained discrepant results. In particular, Roelofsen and FIG. 7. The relationship between (Ca2+ + M&+1Schatzmann found a reactivation with all ATPase activity and the phosphatidyl serine to phospholipids studied in the present paprotein’ratio. The enzyme was partially purified by per, but not with free fatty acids such as differential protein solubilization, isoelectric focus- oleic acid. The experiments reported here, ing, and selective protein incorporation into lipowhich have covered the same lipid-requirsomes. Reconstitution was done at 0°C. ATPase ing parameters by reconstitution techactivity was measured by the procedure described niques, have supported the work of Ronner in Fig. 4. et al. (10). Reactivation of enzyme activity is more pronounced when the lipid phosDISCUSSION phatidyl serine or the fatty acid is present. The effective ability to judge whether or The information in this paper deals with not a membrane enzyme has been purified the reactivation of the human erythrocyte (Ca2+ + MgZ+)-ATPase enzyme by success- typically rests on two criteria: the ability fully reconstituting this protein in a par- to visualize, by gel electrophoresis, an tially purified form in a membrane system increase in the amount of enzyme after with a defined lipid medium. (Caz+ + the isolation procedure, and the increase ENZYME ACTIVITIES DIJBING PARTIAL PIJRIFICATION AND RECONSTITUTION OF THE ERYTHROCYTE ~BMA MEMBRANE Ca2+-ATPase’
RECONSTITUTION
OF THE ERYTHROCYTE
Ca*+-ATPase
209
FIG. 8. Relative protein species distribution during partial purification of the (Ca2+ + Mg2+)-ATPase. Purification was done under the same conditions described in Fig. 7. The membrane protein species distribution for the erythrocyte plasma membrane 0, the Triton X-100.solubilized supematant from the plasma membranes (as), and the phosphatidyl aerinereconstituted system after isoelectric focusing (a) are shown. Analysis of data was by the same procedure described in Fig. 3.
in specific activity of the solution as the enzyme is purified. The first method of determination is difficult to use in this situation because this protein comprises only about 0.02% of the total membrane protein (8). A purification of 500-fold would probably be necessary to visualize this enzyme effectively, and such a purification has yet to be achieved. The second criterion is ambiguous for this particular enzyme. Our results have shown that this (Ca2+ + Me+)-ATPa.se has activities that vary significantly, depending on the amount and type of lipid present. Furthermore, unreported observations in our laboratory have shown that the (Ca2+ + Mg2+)-ATPase is extremely labile with time in Triton X-100. An increase (or even a decrease) in specific activity during a purification step may therefore be a product of three different factors: protein purification, enzyme activation due to the presence of a necessary cofactor, and enzyme inactivation because of protein lability. Therefore, to judge the success of the attempts at purification of this protein, we have chosen to correlate the presence of enzyme activity, regardless of its quantity, with the selective presence of the more readily recognizable protein species of the erythrocyte plasma membrane. This (Ca2+ + Mg2+)-ATPase has been reported by Knauf et al. (8) to have a molecular weight of approximately 150,000, and it should be stressed that an observation of the enrichment in bands of any other molecular weight is not necessary to be construed as a statement of association between the ATPase and that band. In this paper, we have demonstrated
several methods of partially purifying a membrane enzyme. The partial purikation of erythrocyte plasma membrane proteins by selective solubilization with detergent has already been reported by Wolf and Gietzen (19). Selective protein incorporation into liposomes occurs when the supernatant has the detergent removed in the presence of phosphatidyl serine, and the resulting liposomes are separated from free proteins. Isoelectric focusing separates proteins on the basis of their isoelectric points (20). If membrane proteins which have been solubilized in a nonionic detergent such as Triton X-100 are used, these proteins can be separated on the basis of charges that they show while in the micelle. The most noticeable contribution of isoelectric focusing to protein was the decrease of band 3 in the fraction which contained (Ca2+ + Mg2+)-ATPase activity. The dominant protein species after this series of partial purifkations are bands 4.5, 6, and 7. It must be noted that the total (Ca2+ + Mg2+)-ATPase activity in this optimal system is only 9% of that found in the supernatant of the Triton-solubilized plasma membranes (Table III). The enzyme has been found to be inactivated by this detergent with time, and it is assumed that this is the reason for the low yield. In fact, 89% of the total activity in the original supernatant, maintained at 4°C for 24 h in 0.3% Triton X100, is found after isoelectric focusing. This suggests that almost all of the activity of the enzyme in the debilitating environment of the detergent has been accounted for. It is possible, therefore, that much of the go-fold increase in specific
210
PETERSON, RONNER, AND CARAFOLI
activity over this supernatant control by the phosphatidyl serine liposomal system represents a true purification. Because of the properties of the acidic lipid present, it is di&ult to measure Ca2+ transport in the enzymatically active reconstituted systems. These lipids bind calcium ions, which in turn induce transition temperature-like phase changes in the lipid hydrocarbon region and vesicle fusion (21). Work is presently under way to develop a suitable lipid mixture that will promote both enzyme activity and Ca2+ transport. The isoelectric focusing procedure is not yet refined, as witnessed by the large pH range that must be collected to reconstitute an active membrane enzyme. The results reported here were done on small focusing columns, which were more advantageous than a larger column with respect to the amount of focusing components required and the running times of the systems. The major limitation of such a system is that only small amounts of membrane material may be used, thus making the degree of resolution of the experiments less precise. Work is presently under way to adapt the procedures that were used in these small columns to larger systems. ACKNOWLEDGMENTS The authors would like to thank Dr. M. Miiller, Service for Electron Microscopy, ETH Zurich, for preparing the electron microscopy pictures and the Blutspendezentrum Limmattal for supplying the human red blood cells. This project was funded by a Swiss National Science Foundation grant (No. 3.592-0.75) and by a Forschungsprojekt, ETH (No. 0.330.076.60/5). REFERENCES 1. WEED, R. I., AND CHAILLEY, B. (1973) in Red
Cell Shape (Bessis, M., Weed, R. I., and
Leblond, P. F., eds.), pp. 55-68, SpringerVerlag, Berlin/New York. 2. SCHATZMANN,H. J. (1975) in Current Topics in Membranes and Transport (Banner, F., and Kleinzeller, A., eds.), Vol. 6, pp. 125-168, Academic Press, New York. 3. S%XIATZMANN, H. J., AND VINCENZI, F. F. (1969). J. Physiol. (London) 201, 369-395. 4. SCHATZMANN,H. J. (1966) Experientia 22, 364368. 5. WINS, T., AND S~HOFFENIEL~, E. (1966)B&him. Biophys. Actu 120, 341-350. 6. SCHATZMANN, H. J., AND ROSSI, G. L. (1971) B&him. Biophys. Acta 241, 379-392. 7. WOLF, H. U. (1972)B&him. Biophys. Actu 266, 361. 8. KNAUF, P. A., Paovsamo, F., AND HOFFMAN,J. F. (1974) J. Gen. Physiol. 63, 324-336. 9. WOLF, H. U., Dnvxvoss, G., AND LICHTNEB, R. (1976)Experientia 32, 776. 10. R~NNEB, P., GAZZO~I, P., AND CARAFOLI, E. (1977) Arch. Biochem. Biophys. 179, 578-583. 11. ROEMFBEN, B., AND SCHATZMANN, H. J. (1977) B&him. Biophys. Actu 464, 17-36. 12. BEHNKE, J. N., DAGHER, S. M., MAEBEY, T. H., AND DEAL, W. C. (1975) Anal. Biochem. 69, l-9. 13. LOWRY, 0. H., ROSEBIKBUGH,N. J., FAIR, A. L., AND RANDALL, R. J. (1951) J. Bid Chem. 193, 265-275. 14. LOWRY, 0. H., ROBERTS, N. R., LEINER, K. Y., WV, M., AND FARR, A. L. (1954) J. Biol. Chem. 207, 1-18. 15. MEI~~NER, G., CONNER, G. E., AND FLEIBCHER, S. (1973) B&him. Biophys. Actu 298, 246269. 16. STECK,T. L. (1974) J. Cell Bid. 62, l-19. 17. HOLLOWAY, P. W. (1973) And. Biochem. 53, 304-308. 18. GEBICKI, J. M., AND Hnxs, M. (1976) Chcm. Phys. Lipids 16, 142-160. 19. WOLF, H. U., AND GIETZEN, K. Z. (1974)PhysioZ. Chem. 356, 1272. 20. VESTJCBBEBG, 0. (1971) Methods Enzymol. 22, 389-412. 21. HAUSER, H., FINER, E. G., AND DARKE, A. (1977) Biochem. Biophys. Res. Commun. 76,267-274.