[76]
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699
[76] R e c o n s t i t u t i o n of M e m b r a n e P r o c e s s e s
By EFRAIMRACKER Resolution and reconstitution is the approach of classic biochemistry to the analysis of multienzyme systems. With soluble systems such as glycolysis or the oxidative and reductive pentose phosphate cycles, this required the isolation of all the participating catalytic components, which were then simply mixed in appropriate concentrations. The study of multienzyme systems such as oxidative phosphorylation or photophosphorylation, which are embedded in membranes, is more complicated because (a) the firm associations between some of the proteins with each other and with phospholipids make the resolution more difficult, and (b) reconstitution requires formation of a compartment with appropriate physical and chemical properties. Perhaps the most important lesson to be learned from past experience with resolution and reconstitution of membrane proteins is that the isolation of the components must be carried out under conditions that preserve their ability to integrate within the catalytic community of the membrane. A most striking example of how small differences in the isolation procedure determine whether an enzyme is reconstitutively active is succinate dehydrogenase. 1 A catalytically active enzyme isolated in the absence of succinate is reconstitutively incompetent, whereas the same purification performed in the presence of substrate yields a reconstitutively active preparation. Some membrane proteins that have been isolated in the past, based on an assay of either a catalytic or a spectral property, are reconstitutively active; others are not. For example, in the case of cytochrome oxidase, which has been favored with almost as many purification procedures as there are laboratories studying it, the deciding factor turned out to be the detergent that was used. Enzyme preparations isolated with cholate as detergent are reconstitutively active; those isolated with deoxycholate are poor or inactive in reconstitution experiments, z What are the differences between reconstitutively active and inactive proteins? How can one avoid falling into the trap of isolating an inactive preparation ? In the case of succinate dehydrogenase the ability to restore succinoxidase activity to depleted membranes can be restored to a defective T. E. K i n g , J. Biol. Chem. 238, 4 0 3 7 (1963). z R. C a r r o l l a n d E. R a c k e r , J. Biol. Chem. 252, 6981 (1977). METHODS IN ENZYMOLOGY, VOL. LV
Copyright ~ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8
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RECONSTITUTION
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enzyme by incorporation of additional nonheme iron. 3 In the case of CF1, the spinach chloroplast coupling factor, and bacterial F1, a defective enzyme can be repaired by addition of one of its missing subunits which is required for attachment to the membrane. 4"5 In many other instances, such as cytochrome oxidase, the chemistry is unknown and a challenging problem. The answer to the second question is simple. If one uses an assay that depends on the reconstitution of the protein into a membrane, purification always yields a reconstitutively active preparation. This is sometimes easier said than done. Yet in the experience of this writer, efforts spent in the design of such an assay pay off in the long run. They have yielded reconstituted systems of oxidative phosphorylation and photophosphorylation.n Reconstitution assays can sometimes be performed with remarkably crude preparations. For example, an assay for the adenine nucleotide transporter was developed starting with a crude extract of submitochondrial particles. 7 The Pi transporter has been purified from a mitochondria based on a reconstitution assay that can be applied to submitochondrial particles, s I have used intact Halobacterium halobium to incorporate bacteriorhodopsin into liposomes, resulting in a reversal of the direction of proton translocation. The second lesson to be learned from past reconstitution experiments is to keep the approach as broad as possible. Each membrane protein responds differently to the various reconstitution procedures, and at present the approach is entirely empirical. The purpose of this introductory article is to describe the reconstitution procedures currently used in our laboratory and to point out their advantages and disadvantages. I want to emphasize, particularly in view of the somewhat different outlook of Meissner and Fleischer, 8 that reconstitution as described in this article is not an approach to mimic physiological conditions. Reconstitution into artificial liposomes with some 5- to 100-fold excess of phospholipids is used solely for two purposes: (a) for assays during purification of membranous proteins and (b) for the study of the mech3 M. L. B a g i n s k y and Y. Hatefi, J. Biol. Chem. 244, 5313 (1969). 4 H. M. Younis, G. D. Winget, and E. Racker, J. Biol. Chem. 252, 1814 (1977). 5 j. B. Smith and P. C. Sternweis, Biochem. Biophys. Res. Commun. 62, 764 (1975). e E. Racker, " A N e w L o o k at M e c h a n i s m s in Bioenergetics." A c a d e m i c Press, N e w York, 1976. r H. Shertzer and E. Racker, J. Biol. Chem. 251, 2446 (1976). s R. Banerjee, H. Shertzer, B. K a n n e r , and E. Racker, Biochem. Biophys. Res. Commun. 75, 772 (1977). sa G. M e i s s n e r and S. Fleischer, J. Biol. Chem. 249, 302 (1974).
[76]
RECONSTITUTION OF MEMBRANEPROCESSES
701
anism of the catalytic process and for the identification of the participating components. Cholate-Dialysis
(Detergent-Dialysis)
Procedure 9
This was the first procedure used for the reconstitution of oxidative phosphorylation. 9-1' The basic procedure is to mix a suspension of phospholipids that were exposed to sonication in the presence of sodium cholate with the isolated protein at appropriate cholate concentrations (1-3%); this is followed by the slow removal of the detergent by dialysis for about 20 hr. Vesicles formed by this procedure catalyze various processes associated with oxidative phosphorylation such as 32Pi-ATP exchange, 9 electron-transport-linked phosphorylation, '° and ATP-dependent translocation of p r o t o n s . " The cholate-dialysis method is simple and has been used for a variety of membrane systems, such as the reconstitution of the Ca 2+ pump of sarcoplasmic reticulum TM and of the Na + pump of the plasma membrane from a variety of sources such as dog kidney, 13 salt gland of the dogfish, 14 and electric eel. 15 Pure phospholipids can be substituted for the crude mixture. Thus far the most frequently encountered pattern is one in which a ratio of 4:1 for phosphatidylethanolamine/phosphatidylcholine gives optimal results. Addition of small amounts of an acidic phospholipid is advantageous in some cases, but an excess of acidic lipids sometimes inhibits. There are exceptions. In the case of mitochondrial transhydrogenase, phosphatidylethanolamine inhibited and optimal results were obtained with phosphatidylcholine alone.'6 There is a report that the Ca ~+ pump of sarcoplasmic reticulum can be reconstituted by the cholate-dialysis procedure with phosphatidylcholine as the sole phospholipid. ~7 In our hands, this lipid reconstituted the ATPase activity of delipidated enzyme but not the transport activity? 8 Yet experience has taught us to respect small vari9 y. Kagawa and E. Racker, J. Biol. Chem. 246, 5477 (1971). 10E. Racker and A. Kandrach, J. Biol. Chem. 248, 5841 (1973). " Y. Kagawa, A. Kandrach, and E. Racker, J. Biol. Chem. 248, 676 (1973). ,z E. Racker, J. Biol. Chem. 247, 8198 (1972). ,3 S. M. Goldin and S. W. Tong, J. Biol. Chem. 249, 5907 (1974). 14S. Hilden, H. M. Rhee, and L. E. Hokin, J. Biol. Chem. 249, 7432 (1974). '~ E. Racker and L. W. Fisher, Biochem. Biophys. Res. Commun. 67, 1144 (1975). 16j. Rydstr6m, N. Kanner, and E. Racker, Biochem. Biophys. Res. Commun. 67, 831 (1975). 17G. B. Warren, D. A. Toon, N. J. M. BirdsaU, A. G. Lee, and J. C. Metcalfe, Proc. Natl. Acad. Sci. U.S.A. 71,622 (1974). ,s A. F. Knowles, E. Eytan, and E. Racker, J. Biol. Chem. 251, 5161 (1976).
702
RECONSTITUTION
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ations in techniques that may account for discrepancies in results observed by competent investigators. Indeed it may be of value to explore the cause of such divergent findings. The cholate-dialysis procedure is more accurately described as a detergent-dialysis procedure. In some instances "cholate alone is not effective, and mixtures of detergents are required for reconstitution. The Ca 2+ pump of sarcoplasmic reticulum required a mixture of cholate and deoxycholate for optimal transport. TM I have observed that these two detergents also gave higher values for proton translocation of bacteriorhodopsin vesicles that were reconstituted by the dialysis procedure. Similar observations were recorded on the reconstitution of the DCCDsensitive ATPase from thermophilic bacteria. 2° Among the disadvantages of the dialysis procedure is the prolonged exposure to detergent, which is not always well tolerated by membrane proteins. In the reconstitution of oxidative phosphorylation, protective agents such as dithiothreitol and methanol had to be included in the dialysis fluid. One of the reasons for the need for such excessive amounts of phospholipids is that the protein must be protected against the detergent. It was found 2 that the phospholipid concentration can be reduced by a factor of 5 when the exposure time to cholate is significantly reduced by a more rapid reconstitution and dialysis procedure. The apparent specificity of phospholipid requirements may at times be an expression of such a protection mechanism rather than of intrinsic requirements of the catalytic process. Such an interpretation seems to be warranted in view of the observation that different reconstitution procedures are optimized with different mixtures of phospholipids. Another disadvantage of the cholate-dialysis procedure is the time factor. During fractionation of membranous proteins, e.g., on molecular sieve columns, a simple and rapid method of analysis is of great advantage, particularly when the proteins are labile. Although in many instances dialysis for 20 hr gave optimal results, prolonged dialysis (up to 4 days) appeared to be required with some systems, e.g., the sodium pump. 13 More rapid procedures for the dialysis of cholate have been successfully used in such cases. 21 We have used a Sephadex column equilibrated with a low concentration of cholate for the same purpose. 1~ However, too rapid removal through a detergentfree Sephadex column has yielded inactive preparations. A summary of data obtained with proteoliposomes reconstituted by the detergent-dialysis procedure is given in Table I. 1~ A. F. K n o w l e s a n d E. Racker, J. Biol. Chem. 250, 3538 (1975). 20 N. Sone, M. Yoshida, H. Hirata, and Y. Kagawa, J. Biol. Chem. 250, 7917 (1975). 2~ K. J. S w e a d n e r and S. M. Goldin, J. Biol. Chem. 250, 4022 (1975).
[76]
RECONSTITUTION OF MEMBRANEPROCESSES
703
TABLE I ACTIVITIESOF SYSTEMSRECONSTITUTEDBY T H E CHOLATE-DIALYSlSPROCEDURE Reconstituted system
Activitya
32PwATP exchange (mitochondrial) Oxidative phosphorylation site I Oxidative phosphorylation site II plus III Oxidative phosphorylation site III Ca~+pump of sarcoplasmic reticulum Cytochrome oxidase vesicles Proton pump of Halobacterium halobium Na÷ pump with ATPase from Squalus
100-150 nmol/min/mg protein P/O of 0.3-0.5 P/O of 0.4-0.6 P/O of 0.3-0.5 0.6-0.8 txmol/min/mg protein Respiratory control ratio of 8-12 0.5-2/zmol/min/mg protein 5-12 nmol/min/mg protein
acanthias 14
a All values are for activities at 25° (or room temperature).
Detergent-Dilution
P r o c e d u r e 22
During studies of the time course of the reconstitution of c y t o c h r o m e oxidase vesicles, ~3 it was noted that simple dilution of a reconstitution mixture containing about 0.8% cholate lowered the concentration of the detergent sufficiently to allow the assay to be performed. This dilution procedure was extended to the reconstitution of a number of other systems including oxidative phosphorylation. 22 The procedure is rapid since it usually requires only a short period of incubation (10-20 min) before dilution. It was of particular value for the many reconstitution assays performed during fractionation of the oligomycin-sensitive ATPase complex by sucrose gradient centrifugation in the presence of a detergent. 24 In most instances the optimal cholate concentration during the mixing of phospholipids and proteins varies between 0.5 and 0.8%. At least 20-fold dilution is required for assay following the incubation period. An interesting application of the cholatedilution procedure was reported ~5 for the asymmetric incorporation of phage M13 coat protein of E s c h e r i c h i a c o l i into synthetic liposomes. The most striking example of the usefulness of the cholate-dilution procedure was encountered during attempts to reconstitute the DCCDsensitive ATPase complex of spinach chloroplasts. In this case successful reconstitution was achieved by the cholate-dilution procedure, whereas 22 E. Racker, T.-F. Chien, and A. Kandrach, FEBS Lett. 57, 14 (1975). 23 E. Racker, J. Membr. Biol. 10, 221 (1972). ~4R. Serrano, B. I. Kanner, and E. Racker, J. Biol. Chem. 251, 2453 (1976). 25 W, Wickner, Proc. Natl. Acad. Sci. U.S.A., 73, 1159(1976).
704
RECONSTITUTION
[76]
all previous attempts to use the cholate-dialysis procedure had failed to yield active and reproducible results, z6 A serious disadvantage of the cholate-dilution procedure is that it has not been applicable to several systems that have been successfully reconstituted by the cholate-dialysis procedure. For example, bacteriorhodopsin has thus far failed to respond to the dilution procedure, although several detergents have been tested. However, the method has not been systematically explored, and mixtures of appropriate detergents may eventually make this procedure more widely applicable. Systems that are more resistant to high concentrations of detergents, such as the protein obtained from thermophilic bacteria, 2° may tolerate and indeed require more potent detergents for reconstitution. Finally, an interesting aspect of the cholate-dilution procedure is that distinct differences in phospholipid requirements have been encountered. For example, for the a2P~-ATP exchange catalyzed by mitochondrial oligomycin-sensitive ATPase, the optimal mixture of phosphatidylethanolamine/phosphatidylcholine is 1:1,22 whereas it is 4: I for the cholatedialysis procedure. 11 As mentioned earlier this may be indicative of a protective action of phosphatidylethanolamine against cholate. Indeed, marked variations in stability, depending on the phospholipid composition, have been encountered during the early reconstitutions of the respiratory chain. 27 Data obtained with some membrane proteins reconstituted by the cholate-dilution procedure are summarized in Table II. Sonication Procedure a. W i t h o u t F r e e z e - T h a w . ~s This procedure was first developed for the reconstitution of membrane proteins in the absence of detergents. Preparations of the CaZ+-ATPase from sarcoplasmic reticulum, which had been passed through a Sephadex column to remove residual deoxycholate, proved to be refractory to reconstitution by the cholate-dialysis procedure unless deoxycholate was added. TM Yet the same preparation of detergent-depleted ATPase, when mixed with phospholipids and exposed to sonic oscillation, yielded vesicles that were active in Ca ~+ transport, z9 Several other membrane proteins, including bacteriorhodopsin, 2a yielded much more active proteoliposomes by the sonication procedure than by cholate-dialysis. 3° In the case of the Na ÷, K+-ATPase from electric eel,
26G. D. Winget, N. Kanner, and E. Racker, Biochim. Biophys. Acta 460, 490 (1977). 27A. Bruni and E. Racker, J. Biol. Chem. 243, 962 (1968). zs E. Racker, Biochem. Biophys. Res. Commun. 55, 224 (1973). 29E. Racker and E. Eytan, Biochem. Biophys. Res. Commun. 55, 174 (1973). 3oE. Racker and W. Stoeckenius,J. Biol. Chem. 249, 662 (1974).
[76]
RECONSTITUTION OF MEMBRANE PROCESSES
705
T A B L E II ACTIVITIES OF SYSTEMS RECONSTITUTED BY THE DETERGENT-DILUTION PROCEDURE Reconstituted s y s t e m 32P~-ATP e x c h a n g e (mitochondrial ATPase) 3zP~-ATP e x c h a n g e (chloroplast ATPase) Oxidative phosphorylation site III Oxidative phosphorylation site II plus III Ca 2+ p u m p of sarcoplasmic reticulum
Activity ~ 120-160 nmol/min/mg protein 200-400 nmol/min/mg protein P/O of 0.2-0.4 P/O of 0.2-0.4 0.4-0.5 ~,mol/min/mg protein
" All values are for activities at 25 ° (or room temperature).
sonication was in fact the only reconstitution procedure that yielded active vesicles. 1~ A comparison of the data in Tables I and III shows that the rate of Na ÷ translocation by sonicated vesicles with electric eel ATPase is an order of magnitude faster than that by vesicles prepared with dogfish ATPase by the cholate-dialysis procedure. In the case of electric eel ATPase, cholate-dialysis yielded vesicles with rates even below those recorded in Table I for the dogfish ATPase. As can be seen from Table III the rates of Ca 2+ translocation by proteoliposomes obtained by sonication are about one-half of those observed with vesicles obtained by cholate-dialysis, but manipulations of the phospholipid composition 29 yield vesicles with Ca 2+ translocation rates approaching those obtained by the dialysis procedure. It should be emphasized once again that the lipid requirements for
T A B L E III ACTIVITIES OF SYSTEMS RECONSTITUTED BY SONICATION PROCEDURE A Reconstituted s y s t e m azPi-ATP e x c h a n g e Oxidative phosphorylation site II plus III Oxidative phosphorylation site III Ca z+ p u m p of sarcoplasmic reticulum C y t o c h r o m e oxidase vesicles Proton pump of Halobacterium halobium N a ÷ p u m p with electric eel A T P a s e Adenine nucleotide transporter P~ (mitochondrial) transporter
Activity a 50-80 nmol/min/mg protein P/O of 0.2-0.4 P/O of 0.2-0.4 0.3-0.4 txmol/min/mg protein Respiratory control ratio of 3-5 2 - 6 / x m o l / m i n / m g protein 0 . 2 - 0 . 3 / x m o l / m i n / m g protein (at 30 °) 0 . 6 - 0 . 8 / x m o l / m i n / m g protein 0.6-0.8 ~ m o l / m i n / m g protein
a Unless specified otherwise all values are for activities at 25 ° (or room temperature).
706
RECONSTITUTION
[76]
Ca 2+ translocation vary significantly with the reconstitution procedure. With the dialysis procedure, rapid pumping rates were obtained with phosphatidylethanolamine as the only phospholipid. With the sonication procedure, both phosphatidylethanolamine and phosphatidylcholine were required, and supplementation with cardiolipin gave a further increase in the rate. 2a It is possible that in the dialysis procedure small amounts of residual cholate or deoxycholate substitute for the acidic phospholipids. The major advantages of the sonication procedure are that it is rapid and requires no detergent. The method is therefore particularly suitable for proteins that are sensitive to detergents. A major disadvantage is that it is difficult to reproduce the power output of the sonicator. Our experience with probe-type sonicators has been bad, particularly when small volumes have been sonicated. Local heating is difficult to control. The most reproducible results have been obtained with a rather inexpensive bath-type sonicator with a round container (special cylindrical ultrasonic tank and generator, Laboratory Supplies Company, Inc., 29 Jefry Lane, Hicksville, New York 11801). With this instrument it is possible to sonicate small volumes (0. I-0.2 ml) in test tubes of uniform size and dimensions at a controlled temperature. For example, for prolonged sonication (e.g., needed for the Na +, K+-ATPase of the plasma membrane of the electric eel), the bath temperature was kept constant at 4°. 15 On the other hand, for sonication of highly saturated proteoliposomes, e.g., dipalmitoylphosphatidylcholine, the temperature was maintained above the transition temperature of 42°. 31 Rigid adherence to experimental details such as volume, geometry and thickness of the glassware, temperature, water level, and power output is necessary for reproducible results. Time of sonication is, of course, the most critical feature and must be determined with each protein that is being reconstituted. Whereas the Na +, K+-ATPase tolerates reasonably well the exposure to sonication for 30 min, mitochondrial and chloroplast ATPase cannot be sonicated longer than 6 min under comparable conditions. Cytochrome oxidase activity is markedly diminished even after 4-5 min of sonication. Other reconstitution procedures are therefore preferable in the case of cytochrome oxidase. In some instances, e.g., with the Pi transporter of mitochondria, the period of sonication has a sharp time optimum of about 7 min under defined conditions, s In such cases small variations in technique result in marked changes in activity. The appearance of the suspension can at times be used for guidance. For example, considerable inactivation of the Pi transporter as well as the adenine nucleotide transporter has taken place by the time the suspension of phospholipid becomes translucent. 31 E. Racker and P. C. Hinkle, J. Membr. Biol. 17, 181 (1974).
[76]
RECONSTITUTION OF MEMBRANE PROCESSES
707
Thus, sonication is stopped before complete clarification of the suspension is achieved. The rates of some proteoliposomes reconstituted by sonication procedure a are summarized in Table III. b. With F r e e z e - T h a w . 32 This new method of reconstitution was first used for the preparation of proteoliposomes with the glucose carrier from red blood cells. 32 It consists of preparing liposomes by sonication and then adding the protein. The mixture is then quickly frozen by immersing the test tube in a Dry Ice-acetone bath. After thawing at room temperature, the mixture is exposed to brief periods of sonication as in procedure a, but usually for less than 2 rain. In the case of glucose transport optimal sonication time is 20 sec; for the Na+,K+-ATPase of electric eel it is 1 min; and for bacteriorhodopsin it is about 2 rain. The exact time for optimal results varies with the volume, geometry of the test tube, power output, etc. The greatest advantage is that the method can be used for proteins that are sensitive to sonication, such as cytochrome oxidase, without much loss of enzymatic activity. In the case of the Na+,K +ATPase of the electric eel, the optimal time by procedure a is 30 min as compared to 1 rain by procedure b, and the rates of Na + transport by the latter procedure are significantly higher. 33 The highest rates obtained by procedure a were about 300 nmol/min per milligram (at 30°), whereas rates of about 1000 nmol (at 37°) were obtained by the freeze-thaw procedure. In the case of reconstitution of bacteriorhodopsin with the mitochondrial ATPase the rates by the two procedures are similar (E. Racker, unpublished). Some of the rates obtained with proteoliposomes reconstituted by procedure b are given in Table IV. Incorporation Procedure a. With Detergents. 34 In this procedure preformed liposomes are exposed to dilute solutions of membrane proteins at 0° for several hours in the presence of very small amounts of detergent. This method was originally devised in an attempt to obtain insight into the process of membrane biogenesis. Lysolecithin was therefore used as a preferential detergent. However, low concentrations of cholate (0.1%) were equally effective in some systems. Liposomes made with soybean phospholipids and 10% lysolecithin were used in the experiments recorded in Table V. 32 M. K a s a h a r a and P. C. Hinkle, Proc. Natl. Acad. Sci. U.S.A. 73, 396 (1976). 33 D. Cohn and E. Racker, unpublished experiments. 34 G. Eytan, M. J. Matheson, and E. Racker, FEBS Lett. 57, 121 (1975).
708
RECONSTITUTION
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TABLE IV ACTIVITIES OF SYSTEMS RECONSTITUTED BY FREEZE-THAW SONICATION PROCEDUREB Reconstituted system 32pi-ATP exchange Cytochrome oxidase Ca 2+ pump of sarcoplasmic reticulum Proton pump of Halobacterium halobium Photophosphorylation with Halobacterium halobium and mitochondrial ATPase Na ÷ pump with electric eel ATPase
Activity a 70 nmol/min/mg protein Respiratory control ratio of 4 0.4/~mol/min/mg protein 4 p,mol/min/mg protein 50 nmol/min/mg protein 0.8-1.2 p.mol/min/mg protein (at 37°)
a Unless specified otherwise all values are for activities at 250 (or room temperature).
b. Without Detergents. a5 Incorporation of externally added protein to
preformed liposomes takes place without detergents, provided that the appropriate mixture of phospholipids is used to prepare the liposomes. The key feature is the requirement for acidic phospholipids. Optimal concentrations must be titrated in each case. With cytochrome oxidase the optimal amount of phosphatidylserine was 30% (of the total phospholipids), that of phosphatidylinositol was 20-30%, and that of cardiolipin was 10%. In contrast to procedure a, incorporation was very rapid and was completed in the presence of Mg z+ within 2-3 min at room temperature. It was somewhat slower in the absence of Mg 2+ and the presence of EDTA (5-8 min), and it was considerably slower at lower temperatures. Incorporation by procedure b was very sensitive to detergents such as lysolecithin or cholate, which inhibited at very low concentrations. The most important feature of the incorporation procedure is that the proteins that have thus far been examined oriented themselves unidirectionally whereas, with the other reconstitution procedures, proteoliposomes with proteins oriented in both directions were sometimes obtained. This gave rise to considerable difficulties during early attempts at reconstitution of oxidative phosphorylation, sinde proton movements in both directions resulted in uncoupling, lOThe second feature of interest is that the procedure lends itself to the sequential incorporation of proteins, thus giving information on the effect of one protein already present in the membrane on the incorporation of a second protein. Both synergistic and antagonistic effects between proteins have been observed, a6 There are only limited comparative data on the applicability of incora5 G. D. Eytan, M. J. Matheson, and E. Racker, J. Biol. Chem. 251, 6831 (1976). 36 G. D. Eytan and E. Racker, J. Biol. Chem. 252, 3208 (1977).
[76]
RECONSTITUTION OF MEMBRANE PROCESSES
709
TABLE V ACTIVITIES OF SYSTEMS RECONSTITUTED BY INCORPORATION PROCEDURE A Reconstituted s y s t e m a2P~-ATP e x c h a n g e C y t o c h r o m e oxidase vesicles C o e n z y m e Q - c y t o c h r o m e c reductase Ca 2+ p u m p of sarcoplasmic reticulum
Activity a 50-70 nmol/min/mg protein Respiratory control ratio of 4-10 Respiratory control ratio of 3-5 0 . 1 - 0 . 2 / z m o l / m i n / m g protein
All values are for activities at 25 ° (or room temperature).
poration procedure b to various membrane systems (Table VI). Incorporation has been unsuccessfully tried with the Na+,K+-ATPase of the electric eel and with bacteriorhodopsin. However, it is possible to use bacteriorhodopsin reconstituted by sonication with liposomes containing acidic phospholipids and to then incorporate oligomycin-sensitive ATPase. Such vesicles catalyze light-dependent formation of ATP. 35 F u s i o n P r o c e d u r e 37
Liposomes that contain phosphatidylethanolamine and about 30% of either phosphatidylserine or cardiolipin (but not phosphatidylinositol) fuse rapidly on addition of Ca 2+. The fusion procedure has particular value for the reconstitution of mixtures of proteins that are optimally reconstituted by different procedures. For example, cytochrome oxidase reconstituted into liposomes by the cholate-dialysis or incorporation procedure and exhibiting oxidase activity with a high respiratory control can be fused with vesicles that contain the hydrophobic protein of mitochondria reconstituted by the sonication procedure. 37 Fusion can be measured kinetically by measuring the increase in respiration (loss of respiratory control) when the proton channel of the oligomycin-sensitive ATPase is incorporated by fusion into the same vesicle that contains cytochrome oxidase. Another advantage of the fusion procedure is that it yields larger liposomes than the other reconstitution procedures. By the use of osmotic gradients, fusion between large vesicles and even with planar phospholipid bilayers has been achieved? s,a9
37 C. Miller and E. Racker, J. Membr. Biol. 26, 319 (1976). 3s C. Miller, P. A r y a n , J. Telford, and E. Racker, J. Membr. Biol. 30, 271 (1976). 39 C. Miller and E. Racker, J. Membr. Biol. 30, 283 (1976).
710
[76]
RECONSTITUTION
TABLE VI ACTIVITIES OF SYSTEMS RECONSTITUTED BY INCORPORATION PROCEDURE B
Reconstituted system a2Pi-ATP exchange Cytochrome oxidase QH2-cytochrome c reductase Light-driven ATP generation with bacteriorhodopsin and ATPase
Activitya 60 nmol/min/mg protein Respiratory control ratio of 6-10 Respiratory control ratio of 5-6 30 nmol/min/mg protein
All values are for activities at 25° (or room temperature).
R e g e n e r a t i o n S y s t e m s in R e c o n s t i t u t i o n Most reconstituted systems thus far studied are in rather small liposomes. Intravesicular substrates that are utilized or ions that are exported are thus quickly depleted. To maintain zero-order rates, regeneration systems are required. For example, reconstitution of oxidative phosphorylation or of the Na+,K + pump has thus far been restricted to the unnatural orientation (inside-out) of the membrane systems because the nucleotides cannot penetrate the phospholipid bilayer to reach the active site of the enzyme. With this problem in mind, ion channels that provide regenerating systems have been isolated. The adenine nucleotide and Pi transporter, together with mitochondrial ATPase in the right-side-out orientation, hydrolyze A T P added from the outside and r e m o v e ADP and Pi produced inside (R. Banerjee and E. Racker, unpublished experiments). Another example is the reconstitution of the ATP-driven proton pump of mitochondria or chloroplasts. Although it is possible to demonstrate under exacting conditions (pH 6.25) that ATP-dependent proton translocation takes place, 11 the experiments do not lend themselves to quantitative evaluations and are often complicated by artifacts. Instead, a regenerating system of proton delivery by bacteriorhodopsin in the light allows zero-order measurements o f A T P generation with rates that are linear for at least 40 rain and proportional to the amount of ATPase incorporated. 26,3° When valinomycin and nigericin stimulate respiration in reconstituted c y t o c h r o m e oxidase vesicles, 4° valinomycin not only collapses the membrane potential but also serves as a regenerator of K +, which is required for the K+/H + exchange by nigericin. Thus, methods are available for the sequential reconstitution of pro•op. c. Hinkle, J.-J. Kim, and E. Racker, J.
Biol. Chem.
247, 1338(1972).
[77]
RECONSTITUTION OF MITOCHONDRIAL ATP
SYNTHETASE
711
t e i n s , for t h e f u s i o n o f v e s i c l e s w i t h d i f f e r e n t c o m p o s i t i o n s , a n d f o r t h e i n c o r p o r a t i o n o f t r a n s p o r t e r s t h a t s e r v e as r e g e n e r a t o r s o f s o l u t e s i n s i d e t h e l i p o s o m e s . T h e s e p r o c e d u r e s s h o u l d a l l o w us to p r o g r e s s f r o m t h e r e c o n s t i t u t i o n o f s i m p l e s y s t e m s to the a s s e m b l y o f c o m p l e x m u l t i t r a n s port systems with bidirectional solute movements taking place under controlled conditions.
[77] R e c o n s t i t u t i o n
of ATP Synthetase Mitochondria
from Heart
B y YASUO KAGAWA
Assay Methods
Principle. Oligomycin- or DCCD-sensitive mitochondrial ATPase (F0-F1) is composed of a catalytic moiety (F0 and a hydrophobic moiety (F0), which renders F1 sensitive to DCCD. ~,2 When F0"F~ is reconstituted into vesicles, Pi-ATP exchange activity and proton accumulation in the vesicles can be observed in the presence of ATP and Mg2+.a'* Unlike F0"F1 of a thermophilic bacterium (see Article [44], this volume or TF0"F1)) F0"F1 of mitochondria is still crude and unstable. 6 The most practical method to date is to reconstitute crude F0"F~ complex, called the ~'hydrophobic protein" fraction, into vesicles by adding phospholipids and to them add components of F0"F1 such as F~ and OSCP (oligomycin-sensitivity-conferring protein), which are partly denatured during reconstitution of the vesicles. Procedures to measure Pi-ATP exchange activity and proton translocation are essentially the same as described in Article [44] of this volume.
Reagents F~ solution,7 2 mg/ml. A suspension of beef heart F~ in 50% saturated ammonium sulfate solution that has been stored at 4° is centrifuged at 15,000 g for l0 min. The wall of the centrifuge tube 1 y. Y. Y. 4 y. N. 6 R. L.
Kagawa and E. Racker, J. Biol. Chem. 241, 2461 (1966). Kagawa, Biochim. Biophys. Acta (MR) 265, 297 (1972). Kagawa and E. Racker, J. Biol. Chem. 246, 5477 (1971). Kagawa, A. Kandrach, and E. Racker, J. Biol. Chem. 248, 676 (1973). Sone, M. Yoshida, H. Hirata, and Y. Kagawa, J. Biol. Chem. 250, 7917 (1975). Serrano, B. I. Kanner, and E. Racker, J. Biol. Chem. 251, 2453 (1976). L. Horstman and E. Racker, J. Biol. Chem. 245, 1336 (1970).
METHODS IN ENZYMOLOGY, VOL. LV
Copyright © 1979 by Academic Press Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8
R E C O N S T I T U T I O N O F M E M B R A N E PROCESSES
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[76] R e c o n s t i t u t i o n of M e m b r a n e P r o c e s s e s
By EFRAIMRACKER Resolution and reconstitution is the approach of classic biochemistry to the analysis of multienzyme systems. With soluble systems such as glycolysis or the oxidative and reductive pentose phosphate cycles, this required the isolation of all the participating catalytic components, which were then simply mixed in appropriate concentrations. The study of multienzyme systems such as oxidative phosphorylation or photophosphorylation, which are embedded in membranes, is more complicated because (a) the firm associations between some of the proteins with each other and with phospholipids make the resolution more difficult, and (b) reconstitution requires formation of a compartment with appropriate physical and chemical properties. Perhaps the most important lesson to be learned from past experience with resolution and reconstitution of membrane proteins is that the isolation of the components must be carried out under conditions that preserve their ability to integrate within the catalytic community of the membrane. A most striking example of how small differences in the isolation procedure determine whether an enzyme is reconstitutively active is succinate dehydrogenase. 1 A catalytically active enzyme isolated in the absence of succinate is reconstitutively incompetent, whereas the same purification performed in the presence of substrate yields a reconstitutively active preparation. Some membrane proteins that have been isolated in the past, based on an assay of either a catalytic or a spectral property, are reconstitutively active; others are not. For example, in the case of cytochrome oxidase, which has been favored with almost as many purification procedures as there are laboratories studying it, the deciding factor turned out to be the detergent that was used. Enzyme preparations isolated with cholate as detergent are reconstitutively active; those isolated with deoxycholate are poor or inactive in reconstitution experiments, z What are the differences between reconstitutively active and inactive proteins? How can one avoid falling into the trap of isolating an inactive preparation ? In the case of succinate dehydrogenase the ability to restore succinoxidase activity to depleted membranes can be restored to a defective T. E. K i n g , J. Biol. Chem. 238, 4 0 3 7 (1963). z R. C a r r o l l a n d E. R a c k e r , J. Biol. Chem. 252, 6981 (1977). METHODS IN ENZYMOLOGY, VOL. LV
Copyright ~ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8
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RECONSTITUTION
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enzyme by incorporation of additional nonheme iron. 3 In the case of CF1, the spinach chloroplast coupling factor, and bacterial F1, a defective enzyme can be repaired by addition of one of its missing subunits which is required for attachment to the membrane. 4"5 In many other instances, such as cytochrome oxidase, the chemistry is unknown and a challenging problem. The answer to the second question is simple. If one uses an assay that depends on the reconstitution of the protein into a membrane, purification always yields a reconstitutively active preparation. This is sometimes easier said than done. Yet in the experience of this writer, efforts spent in the design of such an assay pay off in the long run. They have yielded reconstituted systems of oxidative phosphorylation and photophosphorylation.n Reconstitution assays can sometimes be performed with remarkably crude preparations. For example, an assay for the adenine nucleotide transporter was developed starting with a crude extract of submitochondrial particles. 7 The Pi transporter has been purified from a mitochondria based on a reconstitution assay that can be applied to submitochondrial particles, s I have used intact Halobacterium halobium to incorporate bacteriorhodopsin into liposomes, resulting in a reversal of the direction of proton translocation. The second lesson to be learned from past reconstitution experiments is to keep the approach as broad as possible. Each membrane protein responds differently to the various reconstitution procedures, and at present the approach is entirely empirical. The purpose of this introductory article is to describe the reconstitution procedures currently used in our laboratory and to point out their advantages and disadvantages. I want to emphasize, particularly in view of the somewhat different outlook of Meissner and Fleischer, 8 that reconstitution as described in this article is not an approach to mimic physiological conditions. Reconstitution into artificial liposomes with some 5- to 100-fold excess of phospholipids is used solely for two purposes: (a) for assays during purification of membranous proteins and (b) for the study of the mech3 M. L. B a g i n s k y and Y. Hatefi, J. Biol. Chem. 244, 5313 (1969). 4 H. M. Younis, G. D. Winget, and E. Racker, J. Biol. Chem. 252, 1814 (1977). 5 j. B. Smith and P. C. Sternweis, Biochem. Biophys. Res. Commun. 62, 764 (1975). e E. Racker, " A N e w L o o k at M e c h a n i s m s in Bioenergetics." A c a d e m i c Press, N e w York, 1976. r H. Shertzer and E. Racker, J. Biol. Chem. 251, 2446 (1976). s R. Banerjee, H. Shertzer, B. K a n n e r , and E. Racker, Biochem. Biophys. Res. Commun. 75, 772 (1977). sa G. M e i s s n e r and S. Fleischer, J. Biol. Chem. 249, 302 (1974).
[76]
RECONSTITUTION OF MEMBRANEPROCESSES
701
anism of the catalytic process and for the identification of the participating components. Cholate-Dialysis
(Detergent-Dialysis)
Procedure 9
This was the first procedure used for the reconstitution of oxidative phosphorylation. 9-1' The basic procedure is to mix a suspension of phospholipids that were exposed to sonication in the presence of sodium cholate with the isolated protein at appropriate cholate concentrations (1-3%); this is followed by the slow removal of the detergent by dialysis for about 20 hr. Vesicles formed by this procedure catalyze various processes associated with oxidative phosphorylation such as 32Pi-ATP exchange, 9 electron-transport-linked phosphorylation, '° and ATP-dependent translocation of p r o t o n s . " The cholate-dialysis method is simple and has been used for a variety of membrane systems, such as the reconstitution of the Ca 2+ pump of sarcoplasmic reticulum TM and of the Na + pump of the plasma membrane from a variety of sources such as dog kidney, 13 salt gland of the dogfish, 14 and electric eel. 15 Pure phospholipids can be substituted for the crude mixture. Thus far the most frequently encountered pattern is one in which a ratio of 4:1 for phosphatidylethanolamine/phosphatidylcholine gives optimal results. Addition of small amounts of an acidic phospholipid is advantageous in some cases, but an excess of acidic lipids sometimes inhibits. There are exceptions. In the case of mitochondrial transhydrogenase, phosphatidylethanolamine inhibited and optimal results were obtained with phosphatidylcholine alone.'6 There is a report that the Ca ~+ pump of sarcoplasmic reticulum can be reconstituted by the cholate-dialysis procedure with phosphatidylcholine as the sole phospholipid. ~7 In our hands, this lipid reconstituted the ATPase activity of delipidated enzyme but not the transport activity? 8 Yet experience has taught us to respect small vari9 y. Kagawa and E. Racker, J. Biol. Chem. 246, 5477 (1971). 10E. Racker and A. Kandrach, J. Biol. Chem. 248, 5841 (1973). " Y. Kagawa, A. Kandrach, and E. Racker, J. Biol. Chem. 248, 676 (1973). ,z E. Racker, J. Biol. Chem. 247, 8198 (1972). ,3 S. M. Goldin and S. W. Tong, J. Biol. Chem. 249, 5907 (1974). 14S. Hilden, H. M. Rhee, and L. E. Hokin, J. Biol. Chem. 249, 7432 (1974). '~ E. Racker and L. W. Fisher, Biochem. Biophys. Res. Commun. 67, 1144 (1975). 16j. Rydstr6m, N. Kanner, and E. Racker, Biochem. Biophys. Res. Commun. 67, 831 (1975). 17G. B. Warren, D. A. Toon, N. J. M. BirdsaU, A. G. Lee, and J. C. Metcalfe, Proc. Natl. Acad. Sci. U.S.A. 71,622 (1974). ,s A. F. Knowles, E. Eytan, and E. Racker, J. Biol. Chem. 251, 5161 (1976).
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ations in techniques that may account for discrepancies in results observed by competent investigators. Indeed it may be of value to explore the cause of such divergent findings. The cholate-dialysis procedure is more accurately described as a detergent-dialysis procedure. In some instances "cholate alone is not effective, and mixtures of detergents are required for reconstitution. The Ca 2+ pump of sarcoplasmic reticulum required a mixture of cholate and deoxycholate for optimal transport. TM I have observed that these two detergents also gave higher values for proton translocation of bacteriorhodopsin vesicles that were reconstituted by the dialysis procedure. Similar observations were recorded on the reconstitution of the DCCDsensitive ATPase from thermophilic bacteria. 2° Among the disadvantages of the dialysis procedure is the prolonged exposure to detergent, which is not always well tolerated by membrane proteins. In the reconstitution of oxidative phosphorylation, protective agents such as dithiothreitol and methanol had to be included in the dialysis fluid. One of the reasons for the need for such excessive amounts of phospholipids is that the protein must be protected against the detergent. It was found 2 that the phospholipid concentration can be reduced by a factor of 5 when the exposure time to cholate is significantly reduced by a more rapid reconstitution and dialysis procedure. The apparent specificity of phospholipid requirements may at times be an expression of such a protection mechanism rather than of intrinsic requirements of the catalytic process. Such an interpretation seems to be warranted in view of the observation that different reconstitution procedures are optimized with different mixtures of phospholipids. Another disadvantage of the cholate-dialysis procedure is the time factor. During fractionation of membranous proteins, e.g., on molecular sieve columns, a simple and rapid method of analysis is of great advantage, particularly when the proteins are labile. Although in many instances dialysis for 20 hr gave optimal results, prolonged dialysis (up to 4 days) appeared to be required with some systems, e.g., the sodium pump. 13 More rapid procedures for the dialysis of cholate have been successfully used in such cases. 21 We have used a Sephadex column equilibrated with a low concentration of cholate for the same purpose. 1~ However, too rapid removal through a detergentfree Sephadex column has yielded inactive preparations. A summary of data obtained with proteoliposomes reconstituted by the detergent-dialysis procedure is given in Table I. 1~ A. F. K n o w l e s a n d E. Racker, J. Biol. Chem. 250, 3538 (1975). 20 N. Sone, M. Yoshida, H. Hirata, and Y. Kagawa, J. Biol. Chem. 250, 7917 (1975). 2~ K. J. S w e a d n e r and S. M. Goldin, J. Biol. Chem. 250, 4022 (1975).
[76]
RECONSTITUTION OF MEMBRANEPROCESSES
703
TABLE I ACTIVITIESOF SYSTEMSRECONSTITUTEDBY T H E CHOLATE-DIALYSlSPROCEDURE Reconstituted system
Activitya
32PwATP exchange (mitochondrial) Oxidative phosphorylation site I Oxidative phosphorylation site II plus III Oxidative phosphorylation site III Ca~+pump of sarcoplasmic reticulum Cytochrome oxidase vesicles Proton pump of Halobacterium halobium Na÷ pump with ATPase from Squalus
100-150 nmol/min/mg protein P/O of 0.3-0.5 P/O of 0.4-0.6 P/O of 0.3-0.5 0.6-0.8 txmol/min/mg protein Respiratory control ratio of 8-12 0.5-2/zmol/min/mg protein 5-12 nmol/min/mg protein
acanthias 14
a All values are for activities at 25° (or room temperature).
Detergent-Dilution
P r o c e d u r e 22
During studies of the time course of the reconstitution of c y t o c h r o m e oxidase vesicles, ~3 it was noted that simple dilution of a reconstitution mixture containing about 0.8% cholate lowered the concentration of the detergent sufficiently to allow the assay to be performed. This dilution procedure was extended to the reconstitution of a number of other systems including oxidative phosphorylation. 22 The procedure is rapid since it usually requires only a short period of incubation (10-20 min) before dilution. It was of particular value for the many reconstitution assays performed during fractionation of the oligomycin-sensitive ATPase complex by sucrose gradient centrifugation in the presence of a detergent. 24 In most instances the optimal cholate concentration during the mixing of phospholipids and proteins varies between 0.5 and 0.8%. At least 20-fold dilution is required for assay following the incubation period. An interesting application of the cholatedilution procedure was reported ~5 for the asymmetric incorporation of phage M13 coat protein of E s c h e r i c h i a c o l i into synthetic liposomes. The most striking example of the usefulness of the cholate-dilution procedure was encountered during attempts to reconstitute the DCCDsensitive ATPase complex of spinach chloroplasts. In this case successful reconstitution was achieved by the cholate-dilution procedure, whereas 22 E. Racker, T.-F. Chien, and A. Kandrach, FEBS Lett. 57, 14 (1975). 23 E. Racker, J. Membr. Biol. 10, 221 (1972). ~4R. Serrano, B. I. Kanner, and E. Racker, J. Biol. Chem. 251, 2453 (1976). 25 W, Wickner, Proc. Natl. Acad. Sci. U.S.A., 73, 1159(1976).
704
RECONSTITUTION
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all previous attempts to use the cholate-dialysis procedure had failed to yield active and reproducible results, z6 A serious disadvantage of the cholate-dilution procedure is that it has not been applicable to several systems that have been successfully reconstituted by the cholate-dialysis procedure. For example, bacteriorhodopsin has thus far failed to respond to the dilution procedure, although several detergents have been tested. However, the method has not been systematically explored, and mixtures of appropriate detergents may eventually make this procedure more widely applicable. Systems that are more resistant to high concentrations of detergents, such as the protein obtained from thermophilic bacteria, 2° may tolerate and indeed require more potent detergents for reconstitution. Finally, an interesting aspect of the cholate-dilution procedure is that distinct differences in phospholipid requirements have been encountered. For example, for the a2P~-ATP exchange catalyzed by mitochondrial oligomycin-sensitive ATPase, the optimal mixture of phosphatidylethanolamine/phosphatidylcholine is 1:1,22 whereas it is 4: I for the cholatedialysis procedure. 11 As mentioned earlier this may be indicative of a protective action of phosphatidylethanolamine against cholate. Indeed, marked variations in stability, depending on the phospholipid composition, have been encountered during the early reconstitutions of the respiratory chain. 27 Data obtained with some membrane proteins reconstituted by the cholate-dilution procedure are summarized in Table II. Sonication Procedure a. W i t h o u t F r e e z e - T h a w . ~s This procedure was first developed for the reconstitution of membrane proteins in the absence of detergents. Preparations of the CaZ+-ATPase from sarcoplasmic reticulum, which had been passed through a Sephadex column to remove residual deoxycholate, proved to be refractory to reconstitution by the cholate-dialysis procedure unless deoxycholate was added. TM Yet the same preparation of detergent-depleted ATPase, when mixed with phospholipids and exposed to sonic oscillation, yielded vesicles that were active in Ca ~+ transport, z9 Several other membrane proteins, including bacteriorhodopsin, 2a yielded much more active proteoliposomes by the sonication procedure than by cholate-dialysis. 3° In the case of the Na ÷, K+-ATPase from electric eel,
26G. D. Winget, N. Kanner, and E. Racker, Biochim. Biophys. Acta 460, 490 (1977). 27A. Bruni and E. Racker, J. Biol. Chem. 243, 962 (1968). zs E. Racker, Biochem. Biophys. Res. Commun. 55, 224 (1973). 29E. Racker and E. Eytan, Biochem. Biophys. Res. Commun. 55, 174 (1973). 3oE. Racker and W. Stoeckenius,J. Biol. Chem. 249, 662 (1974).
[76]
RECONSTITUTION OF MEMBRANE PROCESSES
705
T A B L E II ACTIVITIES OF SYSTEMS RECONSTITUTED BY THE DETERGENT-DILUTION PROCEDURE Reconstituted s y s t e m 32P~-ATP e x c h a n g e (mitochondrial ATPase) 3zP~-ATP e x c h a n g e (chloroplast ATPase) Oxidative phosphorylation site III Oxidative phosphorylation site II plus III Ca 2+ p u m p of sarcoplasmic reticulum
Activity ~ 120-160 nmol/min/mg protein 200-400 nmol/min/mg protein P/O of 0.2-0.4 P/O of 0.2-0.4 0.4-0.5 ~,mol/min/mg protein
" All values are for activities at 25 ° (or room temperature).
sonication was in fact the only reconstitution procedure that yielded active vesicles. 1~ A comparison of the data in Tables I and III shows that the rate of Na ÷ translocation by sonicated vesicles with electric eel ATPase is an order of magnitude faster than that by vesicles prepared with dogfish ATPase by the cholate-dialysis procedure. In the case of electric eel ATPase, cholate-dialysis yielded vesicles with rates even below those recorded in Table I for the dogfish ATPase. As can be seen from Table III the rates of Ca 2+ translocation by proteoliposomes obtained by sonication are about one-half of those observed with vesicles obtained by cholate-dialysis, but manipulations of the phospholipid composition 29 yield vesicles with Ca 2+ translocation rates approaching those obtained by the dialysis procedure. It should be emphasized once again that the lipid requirements for
T A B L E III ACTIVITIES OF SYSTEMS RECONSTITUTED BY SONICATION PROCEDURE A Reconstituted s y s t e m azPi-ATP e x c h a n g e Oxidative phosphorylation site II plus III Oxidative phosphorylation site III Ca z+ p u m p of sarcoplasmic reticulum C y t o c h r o m e oxidase vesicles Proton pump of Halobacterium halobium N a ÷ p u m p with electric eel A T P a s e Adenine nucleotide transporter P~ (mitochondrial) transporter
Activity a 50-80 nmol/min/mg protein P/O of 0.2-0.4 P/O of 0.2-0.4 0.3-0.4 txmol/min/mg protein Respiratory control ratio of 3-5 2 - 6 / x m o l / m i n / m g protein 0 . 2 - 0 . 3 / x m o l / m i n / m g protein (at 30 °) 0 . 6 - 0 . 8 / x m o l / m i n / m g protein 0.6-0.8 ~ m o l / m i n / m g protein
a Unless specified otherwise all values are for activities at 25 ° (or room temperature).
706
RECONSTITUTION
[76]
Ca 2+ translocation vary significantly with the reconstitution procedure. With the dialysis procedure, rapid pumping rates were obtained with phosphatidylethanolamine as the only phospholipid. With the sonication procedure, both phosphatidylethanolamine and phosphatidylcholine were required, and supplementation with cardiolipin gave a further increase in the rate. 2a It is possible that in the dialysis procedure small amounts of residual cholate or deoxycholate substitute for the acidic phospholipids. The major advantages of the sonication procedure are that it is rapid and requires no detergent. The method is therefore particularly suitable for proteins that are sensitive to detergents. A major disadvantage is that it is difficult to reproduce the power output of the sonicator. Our experience with probe-type sonicators has been bad, particularly when small volumes have been sonicated. Local heating is difficult to control. The most reproducible results have been obtained with a rather inexpensive bath-type sonicator with a round container (special cylindrical ultrasonic tank and generator, Laboratory Supplies Company, Inc., 29 Jefry Lane, Hicksville, New York 11801). With this instrument it is possible to sonicate small volumes (0. I-0.2 ml) in test tubes of uniform size and dimensions at a controlled temperature. For example, for prolonged sonication (e.g., needed for the Na +, K+-ATPase of the plasma membrane of the electric eel), the bath temperature was kept constant at 4°. 15 On the other hand, for sonication of highly saturated proteoliposomes, e.g., dipalmitoylphosphatidylcholine, the temperature was maintained above the transition temperature of 42°. 31 Rigid adherence to experimental details such as volume, geometry and thickness of the glassware, temperature, water level, and power output is necessary for reproducible results. Time of sonication is, of course, the most critical feature and must be determined with each protein that is being reconstituted. Whereas the Na +, K+-ATPase tolerates reasonably well the exposure to sonication for 30 min, mitochondrial and chloroplast ATPase cannot be sonicated longer than 6 min under comparable conditions. Cytochrome oxidase activity is markedly diminished even after 4-5 min of sonication. Other reconstitution procedures are therefore preferable in the case of cytochrome oxidase. In some instances, e.g., with the Pi transporter of mitochondria, the period of sonication has a sharp time optimum of about 7 min under defined conditions, s In such cases small variations in technique result in marked changes in activity. The appearance of the suspension can at times be used for guidance. For example, considerable inactivation of the Pi transporter as well as the adenine nucleotide transporter has taken place by the time the suspension of phospholipid becomes translucent. 31 E. Racker and P. C. Hinkle, J. Membr. Biol. 17, 181 (1974).
[76]
RECONSTITUTION OF MEMBRANE PROCESSES
707
Thus, sonication is stopped before complete clarification of the suspension is achieved. The rates of some proteoliposomes reconstituted by sonication procedure a are summarized in Table III. b. With F r e e z e - T h a w . 32 This new method of reconstitution was first used for the preparation of proteoliposomes with the glucose carrier from red blood cells. 32 It consists of preparing liposomes by sonication and then adding the protein. The mixture is then quickly frozen by immersing the test tube in a Dry Ice-acetone bath. After thawing at room temperature, the mixture is exposed to brief periods of sonication as in procedure a, but usually for less than 2 rain. In the case of glucose transport optimal sonication time is 20 sec; for the Na+,K+-ATPase of electric eel it is 1 min; and for bacteriorhodopsin it is about 2 rain. The exact time for optimal results varies with the volume, geometry of the test tube, power output, etc. The greatest advantage is that the method can be used for proteins that are sensitive to sonication, such as cytochrome oxidase, without much loss of enzymatic activity. In the case of the Na+,K +ATPase of the electric eel, the optimal time by procedure a is 30 min as compared to 1 rain by procedure b, and the rates of Na + transport by the latter procedure are significantly higher. 33 The highest rates obtained by procedure a were about 300 nmol/min per milligram (at 30°), whereas rates of about 1000 nmol (at 37°) were obtained by the freeze-thaw procedure. In the case of reconstitution of bacteriorhodopsin with the mitochondrial ATPase the rates by the two procedures are similar (E. Racker, unpublished). Some of the rates obtained with proteoliposomes reconstituted by procedure b are given in Table IV. Incorporation Procedure a. With Detergents. 34 In this procedure preformed liposomes are exposed to dilute solutions of membrane proteins at 0° for several hours in the presence of very small amounts of detergent. This method was originally devised in an attempt to obtain insight into the process of membrane biogenesis. Lysolecithin was therefore used as a preferential detergent. However, low concentrations of cholate (0.1%) were equally effective in some systems. Liposomes made with soybean phospholipids and 10% lysolecithin were used in the experiments recorded in Table V. 32 M. K a s a h a r a and P. C. Hinkle, Proc. Natl. Acad. Sci. U.S.A. 73, 396 (1976). 33 D. Cohn and E. Racker, unpublished experiments. 34 G. Eytan, M. J. Matheson, and E. Racker, FEBS Lett. 57, 121 (1975).
708
RECONSTITUTION
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TABLE IV ACTIVITIES OF SYSTEMS RECONSTITUTED BY FREEZE-THAW SONICATION PROCEDUREB Reconstituted system 32pi-ATP exchange Cytochrome oxidase Ca 2+ pump of sarcoplasmic reticulum Proton pump of Halobacterium halobium Photophosphorylation with Halobacterium halobium and mitochondrial ATPase Na ÷ pump with electric eel ATPase
Activity a 70 nmol/min/mg protein Respiratory control ratio of 4 0.4/~mol/min/mg protein 4 p,mol/min/mg protein 50 nmol/min/mg protein 0.8-1.2 p.mol/min/mg protein (at 37°)
a Unless specified otherwise all values are for activities at 250 (or room temperature).
b. Without Detergents. a5 Incorporation of externally added protein to
preformed liposomes takes place without detergents, provided that the appropriate mixture of phospholipids is used to prepare the liposomes. The key feature is the requirement for acidic phospholipids. Optimal concentrations must be titrated in each case. With cytochrome oxidase the optimal amount of phosphatidylserine was 30% (of the total phospholipids), that of phosphatidylinositol was 20-30%, and that of cardiolipin was 10%. In contrast to procedure a, incorporation was very rapid and was completed in the presence of Mg z+ within 2-3 min at room temperature. It was somewhat slower in the absence of Mg 2+ and the presence of EDTA (5-8 min), and it was considerably slower at lower temperatures. Incorporation by procedure b was very sensitive to detergents such as lysolecithin or cholate, which inhibited at very low concentrations. The most important feature of the incorporation procedure is that the proteins that have thus far been examined oriented themselves unidirectionally whereas, with the other reconstitution procedures, proteoliposomes with proteins oriented in both directions were sometimes obtained. This gave rise to considerable difficulties during early attempts at reconstitution of oxidative phosphorylation, sinde proton movements in both directions resulted in uncoupling, lOThe second feature of interest is that the procedure lends itself to the sequential incorporation of proteins, thus giving information on the effect of one protein already present in the membrane on the incorporation of a second protein. Both synergistic and antagonistic effects between proteins have been observed, a6 There are only limited comparative data on the applicability of incora5 G. D. Eytan, M. J. Matheson, and E. Racker, J. Biol. Chem. 251, 6831 (1976). 36 G. D. Eytan and E. Racker, J. Biol. Chem. 252, 3208 (1977).
[76]
RECONSTITUTION OF MEMBRANE PROCESSES
709
TABLE V ACTIVITIES OF SYSTEMS RECONSTITUTED BY INCORPORATION PROCEDURE A Reconstituted s y s t e m a2P~-ATP e x c h a n g e C y t o c h r o m e oxidase vesicles C o e n z y m e Q - c y t o c h r o m e c reductase Ca 2+ p u m p of sarcoplasmic reticulum
Activity a 50-70 nmol/min/mg protein Respiratory control ratio of 4-10 Respiratory control ratio of 3-5 0 . 1 - 0 . 2 / z m o l / m i n / m g protein
All values are for activities at 25 ° (or room temperature).
poration procedure b to various membrane systems (Table VI). Incorporation has been unsuccessfully tried with the Na+,K+-ATPase of the electric eel and with bacteriorhodopsin. However, it is possible to use bacteriorhodopsin reconstituted by sonication with liposomes containing acidic phospholipids and to then incorporate oligomycin-sensitive ATPase. Such vesicles catalyze light-dependent formation of ATP. 35 F u s i o n P r o c e d u r e 37
Liposomes that contain phosphatidylethanolamine and about 30% of either phosphatidylserine or cardiolipin (but not phosphatidylinositol) fuse rapidly on addition of Ca 2+. The fusion procedure has particular value for the reconstitution of mixtures of proteins that are optimally reconstituted by different procedures. For example, cytochrome oxidase reconstituted into liposomes by the cholate-dialysis or incorporation procedure and exhibiting oxidase activity with a high respiratory control can be fused with vesicles that contain the hydrophobic protein of mitochondria reconstituted by the sonication procedure. 37 Fusion can be measured kinetically by measuring the increase in respiration (loss of respiratory control) when the proton channel of the oligomycin-sensitive ATPase is incorporated by fusion into the same vesicle that contains cytochrome oxidase. Another advantage of the fusion procedure is that it yields larger liposomes than the other reconstitution procedures. By the use of osmotic gradients, fusion between large vesicles and even with planar phospholipid bilayers has been achieved? s,a9
37 C. Miller and E. Racker, J. Membr. Biol. 26, 319 (1976). 3s C. Miller, P. A r y a n , J. Telford, and E. Racker, J. Membr. Biol. 30, 271 (1976). 39 C. Miller and E. Racker, J. Membr. Biol. 30, 283 (1976).
710
[76]
RECONSTITUTION
TABLE VI ACTIVITIES OF SYSTEMS RECONSTITUTED BY INCORPORATION PROCEDURE B
Reconstituted system a2Pi-ATP exchange Cytochrome oxidase QH2-cytochrome c reductase Light-driven ATP generation with bacteriorhodopsin and ATPase
Activitya 60 nmol/min/mg protein Respiratory control ratio of 6-10 Respiratory control ratio of 5-6 30 nmol/min/mg protein
All values are for activities at 25° (or room temperature).
R e g e n e r a t i o n S y s t e m s in R e c o n s t i t u t i o n Most reconstituted systems thus far studied are in rather small liposomes. Intravesicular substrates that are utilized or ions that are exported are thus quickly depleted. To maintain zero-order rates, regeneration systems are required. For example, reconstitution of oxidative phosphorylation or of the Na+,K + pump has thus far been restricted to the unnatural orientation (inside-out) of the membrane systems because the nucleotides cannot penetrate the phospholipid bilayer to reach the active site of the enzyme. With this problem in mind, ion channels that provide regenerating systems have been isolated. The adenine nucleotide and Pi transporter, together with mitochondrial ATPase in the right-side-out orientation, hydrolyze A T P added from the outside and r e m o v e ADP and Pi produced inside (R. Banerjee and E. Racker, unpublished experiments). Another example is the reconstitution of the ATP-driven proton pump of mitochondria or chloroplasts. Although it is possible to demonstrate under exacting conditions (pH 6.25) that ATP-dependent proton translocation takes place, 11 the experiments do not lend themselves to quantitative evaluations and are often complicated by artifacts. Instead, a regenerating system of proton delivery by bacteriorhodopsin in the light allows zero-order measurements o f A T P generation with rates that are linear for at least 40 rain and proportional to the amount of ATPase incorporated. 26,3° When valinomycin and nigericin stimulate respiration in reconstituted c y t o c h r o m e oxidase vesicles, 4° valinomycin not only collapses the membrane potential but also serves as a regenerator of K +, which is required for the K+/H + exchange by nigericin. Thus, methods are available for the sequential reconstitution of pro•op. c. Hinkle, J.-J. Kim, and E. Racker, J.
Biol. Chem.
247, 1338(1972).
[77]
RECONSTITUTION OF MITOCHONDRIAL ATP
SYNTHETASE
711
t e i n s , for t h e f u s i o n o f v e s i c l e s w i t h d i f f e r e n t c o m p o s i t i o n s , a n d f o r t h e i n c o r p o r a t i o n o f t r a n s p o r t e r s t h a t s e r v e as r e g e n e r a t o r s o f s o l u t e s i n s i d e t h e l i p o s o m e s . T h e s e p r o c e d u r e s s h o u l d a l l o w us to p r o g r e s s f r o m t h e r e c o n s t i t u t i o n o f s i m p l e s y s t e m s to the a s s e m b l y o f c o m p l e x m u l t i t r a n s port systems with bidirectional solute movements taking place under controlled conditions.
[77] R e c o n s t i t u t i o n
of ATP Synthetase Mitochondria
from Heart
B y YASUO KAGAWA
Assay Methods
Principle. Oligomycin- or DCCD-sensitive mitochondrial ATPase (F0-F1) is composed of a catalytic moiety (F0 and a hydrophobic moiety (F0), which renders F1 sensitive to DCCD. ~,2 When F0"F~ is reconstituted into vesicles, Pi-ATP exchange activity and proton accumulation in the vesicles can be observed in the presence of ATP and Mg2+.a'* Unlike F0"F1 of a thermophilic bacterium (see Article [44], this volume or TF0"F1)) F0"F1 of mitochondria is still crude and unstable. 6 The most practical method to date is to reconstitute crude F0"F~ complex, called the ~'hydrophobic protein" fraction, into vesicles by adding phospholipids and to them add components of F0"F1 such as F~ and OSCP (oligomycin-sensitivity-conferring protein), which are partly denatured during reconstitution of the vesicles. Procedures to measure Pi-ATP exchange activity and proton translocation are essentially the same as described in Article [44] of this volume.
Reagents F~ solution,7 2 mg/ml. A suspension of beef heart F~ in 50% saturated ammonium sulfate solution that has been stored at 4° is centrifuged at 15,000 g for l0 min. The wall of the centrifuge tube 1 y. Y. Y. 4 y. N. 6 R. L.
Kagawa and E. Racker, J. Biol. Chem. 241, 2461 (1966). Kagawa, Biochim. Biophys. Acta (MR) 265, 297 (1972). Kagawa and E. Racker, J. Biol. Chem. 246, 5477 (1971). Kagawa, A. Kandrach, and E. Racker, J. Biol. Chem. 248, 676 (1973). Sone, M. Yoshida, H. Hirata, and Y. Kagawa, J. Biol. Chem. 250, 7917 (1975). Serrano, B. I. Kanner, and E. Racker, J. Biol. Chem. 251, 2453 (1976). L. Horstman and E. Racker, J. Biol. Chem. 245, 1336 (1970).
METHODS IN ENZYMOLOGY, VOL. LV
Copyright © 1979 by Academic Press Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8
