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Reconstitution, a way of biochemical research; some new approaches to membrane-bound enzymes
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 198, No. 2, December, pp. 470-477, 1979
Reconstitution,
a Way of Biochemical Research; Some New Approaches to Membrane-Bound Enzyme&
E. RACKER, Section
B. VIOLAND,
of Biochemistry,
Molecular
S. O’NEAL, & Cell
Biology,
M. ALFONZO, Cornell
University,
AND J. TELFORD Ithaca,
New
York
l&953
Received July 30, 1979 A variety of reconstitution procedures has been developed for the analysis of the biological activity of purified membrane proteins. In this paper we describe some new procedures. One is based on the action of two detergents, one ionic and one nonionic. Another method takes advantage of rather unique properties of octylglucoside which, in contrast to the detergents used in the first procedure, allows for virtually instantaneous reconstitution. This procedure is now the method of choice for the reconstitution of bacteriorhodopsin, and the proton pumps of mitochondria or chloroplasts. A third procedure takes advantage of the observation that very short periods of sonication are required when certain solvents (e.g., decane) are present in low concentrations.
In the course of studies on the hexose monophosphate shunt (the Warburg-Dickens pathway), two experiments were performed which illustrate the value of the resolution-reconstitution approach. In crude enzyme preparations from yeast, phosphogluconate was oxidatively decarboxylated with ribose 5-phosphate as a product of the reaction (1). However, with a purified enzyme ribulose 5-phosphate was formed and a second enzyme, an isomerase, was needed for the conversion to ribose li-phosphate (2). An enzyme from yeast which was crystallized and named transketolase (3) appeared to degrade ribulose 5-phosphate to glyceraldehyde 3-phosphate. However, after five to six recrystallizations, transketolase no longer degraded ribulose 5-phosphate and a second enzyme, an epimerase, had to be added to generate xylulose 5-phosphate, the substrate of transketolase (4). When we started to work on oxidative phosphorylation 20 years ago, we were naive enough to expect success with the same approach of resolution and reconstitution. It took about 10 years of frustration before we learned that this approach is 1 Dedicated to Professor Frank Dickens on the occasion of his eightieth birthday. 0003-9861/79/140470-08$02.00/O
Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
470
actually feasible. However, instead of allowing the reactions to take place in solutions as in the case of free-living enzymes, the membrane-bound enzymes had to be provided with special housing facilities. We had to build compartments for them in the form of phospholipid vesicles, called liposomes, which allowed the hydrophobic enzymes to assemble asymmetrically and fulfill their function as ion translocators. During the past 8 years we have developed a variety of methods of reconstitution into liposomes (5). The first, a cholate dialysis procedure (6) is still most widely used for a variety of membranous proteins. But some proteins do not tolerate exposure to cholate for prolonged periods and a sonication procedure was developed which avoided the use of detergents (7). Prolonged exposure to sonication is detrimental for some proteins and a new procedure, freeze-thaw sonication, was developed (8) which minimized the sonication time. A cholate dilution procedure (9) proved to be milder and more rapid than cholate dialysis. However, it is not applicable to some systems, e.g., bacteriorhodopsin, and often requires several hours of incubation. A survey of several other detergents and mixtures of detergents yielded suitable alternative methods which will be described
RECONSTITUTION TABLE EFFECT
OF MEMBRANE-BOUND
I
OF TIME OF INTERACTION BETWEEN CYTOCHROME OXIDASE AND PHOSPHOLIPIDS IN THE PRESENCE OF VARIOUS DETERGENT MIXTURES” Cholate (0.5%)octylglucoside (0.8%)
Cholate (1%) Time of incubation at 0°C
Respiratory control ratio
0 30 min 120 min 3h 20 h
1.0 1.2 1.3 1.7 4.0
Time of Respiratory incubation control at 0°C (min) ratio 0 15 30 90 160
1.0 2.5 3.5 4.5 5.0
a The experimental procedure was described under Materials and Methods. Crude soybean phospholipids were used at a lipid to protein ratio of 25. Cytochrome oxidase was present at a concentration of 1 mg/ml.
here. Particularly, octylglucoside proved most valuable for the reconstitution of bacteriorhodopsin and the proton pumps of mitochondria and chloroplasts. A new sonication procedure in the presence of low concentrations of solvents is also described. MATERIALS
AND METHODS
Preparations Cholic acid was recrystallized as described (6). Octylglucoside was either synthesized (10) or purchased from Calbiochem. Soybean phospholipids (asolectin) were obtained Tom Associated Concentrates, Woodside, New York. Phosphatidylethanolamine and phosphatidylcholine were prepared from bovine heart mitochondria (11). Bacteriorhodopsin (12), cytochrome oxidase (13), Ca*+-ATPase (lo), Na+K+-ATPase (14), and the F, complex of mitochondrial F,F,-ATPase2 (15) were purified as described in the references. Chloroplast ATPase complex was purified as described (16) except that lettuce leaves were used as the source and the extraction was performed with 0.44% octylglucoside ’ Abbreviations used: F,, couplingfactor 1 (ATPase); Fe, a membranous preparation from mitochondria conferring oligomycin (or rutamycin) sensitivity to F,; OSCP, oligomycin sensitivity conferring protein; PC, phosphatidylcholine; PE, phosphatidylethanolamine; Tricine, N-tris(hydroxymethyl)methylglycine.
ENZYMES
471
and 0.1% sodium cholate instead of the higher concentrations used with spinach leaves.
Reconstitution
Procedures
(a) Detmpnt &&ion. Phospholipids were sonicated in a bath-type sonic&or (7) to clarity (10 to 20 min) usually at 40 to 50 mM concentrations. They were mixed with protein, in the presence of detergent, at a phospholipid to protein ratio specified in the legends of tables and figures. After given time periods, samples were taken and diluted at least 20-fold in the enzyme assay mixture. If the sensitivity of the assay did not permit such a dilution, the samples were first diluted with buffer, centrifuged for 30 min at 55,000 rpm (65 Ti rotor in a Spinco centrifuge), suspended in a small volume of buffer, and then assayed. (b) Solvent sonication. Phospholipid suspensions were prepared and mixed with proteins as described under (a). Then a suitable amount of solvent (e.g., 2% 1-bromohexane) was added and the mixture was sonicated 15 to 30 s. The milky appearance of the suspension is caused by the solvent and can be observed in the absence of any phospholipids.
Assay Procedures Respiratory control of cytochrome oxidase (13), proton pumping by bacteriorhodopsin (17), 32P,-ATP exchange (16), Cal+ pump activity (18), and light-driven ATP formation (‘7) were assayed as described in the references. RESULTS
Reconstitution of Cytochrome Oxidase Vesicles with Respiratory Control Respiratory control developed slowly when cytochrome oxidase was incubated at 0°C with soybean phospholipids in the presence of 1% cholate. As shown in Table I, addition of octylglucoside greatly accelerated the reconstitution process. Optimal values were obtained when the concentrations of cholate and octylglucoside were carefully balanced against each other (Figs. 1A and B). The phospholipid to protein ratio in the experiment shown in Table I was 25 (w/w). With some preparations of cytochrome oxidase which were unstable under these conditions, a higher phospholipid to protein ratio was required (Table II). Octylglucoside can be used as the only detergent added at a 1.5% concentration (Table II). This is the most rapid reconstitution procedure ,since respiratory control is established with only a brief incubation of
472
RACKER
ET AL.
1
FIG. 1. (a) Titration of octylglucoside for the reconstitution of cytochrome oxidase vesicles with respiratory control. Asolectin lipsomes at 50 mM were prepared by sonication for 15 min in 50 mM IG', (pH 7.5) as described previously (6). Reconstitutions were performed for 2 h at 0°C in a medium containing 1 mg cytochrome oxidase/ml in the presence of 0.5% sodium cholate and varying amounts of octylglucoside as indicated. The phospholipid to protein ratio was 16:l. Ten microliters was used for each assay. (b) Titration of cholate for the reconstitution of cytochrome oxidase vesicles with respiratory control. The experimental conditions were as described for (a) with 0.7% octylglucoside and varying amounts of cholate as indicated.
the mixture before dilution. As will be apparent from subsequent data, each system has its own optimal condition for reconstitution. In the case of cytochrome oxidase the optimum octylglucoside concentration was close to 1.25% but respiratory control dropped sharply at slightly lower concentrations. A new procedure for the reconstitution of cytochrome oxidase vesicles is described in Table III. The method is a variant of the sonication method based on the observation that the sonication time can be drastically reduced if the liposome-protein mixture is sonicated in the presence of a solvent. A large number of various solvents were
tested and a representative group is listed in the table. Respiratory control ratios over 5 are readily obtained by this method. Reconstitution of the Bacteriorhodopsin Proton Pump Bacteriorhodopsin was readily incorporated into liposomes in the presence of 1.25 to 1.75% octylglucoside. Optimal proton pump activity upon dilution was achieved soon after mixing of the protein and phospholipid. Steady-state proton pumping in the light as well as initial rates of H+ translocation were high (4000 to 5000 n atom H+/ min/mg protein). These rates are consider-
TABLE
II
COMPARISONOFCHOLATE-OCTYLGLUCOSIDEANDOCTYLGLUCOSIDE DILUTION RECONSTITUTION OFCYTOCHROME OXIDASE VESICLES WITHRESPIRATORYCONTROL~ Cholate (0.7%)-octylglucoside
Experiment Experiment
1 2
(0.7%)
Time of incubation at 0°C (min)
Respiratory control ratio
60 60
2.2 4.1
Octylglucoside (1.5%) Time of incubation at 0” (min) 10 10
Respiratory control ratio 1.9 4.6
a The experimental procedure was as described in Table I, except that a different cytochrome oxidase preparation was used and the phospholipid to protein ratio was 25 in Experiment 1 and 40 in Experiment 2.
RECONSTITUTION TABLE
OF IVIEMBRANE-BOUND
III
SOLVENT SONICATION RECONSTITUTION OF CYTOCHROME OXIDASE~
Solvent
Ccl
Time of sonication (set)
1-Bromohexane 1-Bromohexane 1-Bromohexane 1-Bromohexane 1-Bromohexane I-Bromohexane 1-Bromohexane Tetradecane Decane Octane Heptane Hexane Pentane Pentane Toluene Ether Ether
1 1 2 2 3 4 5 4 4
15 30 15 30 15 15 15 15 15
4 4 8 2 2 2
15 15 30 30 15 30
Respiratory control ratio 1.6 2.1 2.3 6.0 3.3 3.8 4.0 2.3 4.9 4.2 4.6 3.3 1.5 1.9 2.2 2.2 2.0
ably higher than in liposomes reconstituted by other procedures such as cholate dialysis (17), direct sonication (7) or freeze-thaw sonication (8). A value for vesicles, obtained by the last procedure under experimental conditions comparable to those used for the octylglucoside dilution method, is shown in Table IV for comparison. Below 1% octylglucoside the reconstitution became ineffective. However, when cholate, which by itself did not facilitate reconstitution, was added to 1% octylglucoside, reconstitution took place. Once again, reconstitution was dependent on the incubation time of the detergent mixture. At 1% octylglucoside-0.25% cholate, it took 5 h incubation at 0°C to attain maximal H+ pumping rate. Reconstitution Chloroplasts Exchange
a In a final volume of 0.1 ml 4 pmol of sonicated asolectin (in the presence of 50 mM KP,, pH 7.5) were mixed in a small test tube (12 x 74 mm) with 120 pg of cytochrome oxidase and 2 to 4 ~1 of solvent. The suspension appeared milky after brief sonication in a water bath sonicator. Respiratory control ratio was determined by measuring oxygen uptake in the absence and presence of valinomycin (2 pg/ml)-nigericin
(2 j@nl). TABLE
473
ENZYMES
of the H+-ATPase from Catalyzing 32Pi-ATP
The best reconstitution of the chloroplast ATPase complex described thus far is the freeze-thaw procedure with no sonication (16). The octylglucoside dilution procedure gave even higher activity and was very reproducible. It is apparent from Fig. 2 that the concentration of octylglucoside required for optimal reconstitution of the 32Pi-ATP exchange varied with the concentration of phospholipids and protein. IV
RECONSTITUTION OF BACTERIORHODOPSIN WITH OCTYLGLUCOSIDE OR OCTYLGLUCOSIDE-CHOLATE MIXTURES’ Experiment Experiment
2
1
Concentration of octylglucoside (%)
n atoms H+/ mg protein
Concentration of cholate (with 1% octylglucoside) (%)
0.5 1.0 1.25 1.50 1.75 2.0 4.2
0 120 702 601 538 394 212
0 0.125 0.250 0.370 0.250 (no octylglucoside) Liposomes prepared by freeze-thaw sonication
n atoms H+/ mg protein 135 725 705 411 0 173
a In a final volume of 40 ~1 containing 75 mM KCl, 1 mM Na-Tricine (pH 8.0), 0.8 pmol of sonicated asolectin was mixed with 10 pg of bacteriorhodopsin in the presence of the indicated concentrations of detergent. The entire sample was added to 1 ml of 0.15 M KC1 for assay.
474
RACKER
ET AL.
Reconstitution of Purified Mitochondrial ATPase Complex and Bacteriorhodopsin Catal yxing ATP Formation As shown in Fig. 3, simultaneous reconstitution of two different functional proteins by the octylglucoside dilution procedure is possible if there is enough overlap with respect to reconstitution conditions for each component. The rates of ATP formation in the light in this experiment were two- to fivefold higher than previously reported for either the cholate dialysis (17) or the sonication (7) procedures. Freeze-thaw sonication (8) also gave lower activity than octylglucoside dilution (Fig. 3). The need for careful titration of octylglucoside with different protein and phospholipid concentrations is illustrated in Fig. 3. It can be seen that the optimal concentration for one mixture may be completely ineffective for another. Since lower concentrations were needed for the chloroplast ATPase than for bacteriorhodopsin, simultaneous incorporation did not yield very active preparations. In such a case sequential reconstitution (5) can be used as will be described below. Reconstitution of Sarcoplasmic Reticulum Ca2+-ATPase Catalyzing Ca2+ Transport As shown in Fig. 4, the purified Ca2+ATPase from sarcoplasmic reticulum was incorporated into liposomes at various concentrations of octylglucoside. It should be noted that 1.2596, the optimal concentration for reconstitution of bacteriorhodopsin (Table III), is virtually ineffective for the Ca*+-ATPase. Experiments similar to those shown in Fig. 4 were performed with sarcoplasmic reticulum instead of purified enzyme with comparable results. Reconstitution Catalyzing
of Na+K+-ATPase Na+ Uptake
As shown in Fig. 5, purified Na+K+ATPase from electric eel was incorporated into liposomes at various concentrations of octylglucoside. It can be seen that higher concentrations of octylglucoside are required compared to reconstitutions of other membrane proteins, but in general the titration is similar to that observed with the Ca*+-
FIG. 2. Reconstitution of chloroplast H+-ATPase catalyzing 32P,-ATP exchange activity. (a) In a final volume of 0.1 ml containing 3.5 mg sonicated asolectin, 80 mM Na-Tricine (pH 8.0), 0.1 mM EDTA, and oetylglucoside as indicated were incubated at 0°C in the presence of 100 pg chloroplast ATPase complex. After 10 min, lo-p1 samples were assayed for 3ZPi-ATP exchange in l-ml reaction mixture. (b) Conditions were as in (a) except that 2 mg sonicated asolectin and 50 pg of the ATPase complex were used.
ATPase (Fig. 4). The ratio of Na+ transport is high compared to that obtained by- the freeze-thaw sonication procedure (D. Cohn,
1 FIG. 3. Reconstitution of mitoehondrial H+-ATPase complex and bacteriorhodopsin catalyzing ATP formation. (a) In a final volume of 0.1 ml, 0.625 pmol sonicated PE/PC (1:l molar ratio), 10 mM Na-Tricine (pH 8.0), 10 mM KCl, and octylglucoside as indicated were incubated at 0°C in the presence of 80 pg bacteriorhodopsin and 32 pg of mitochondrial F,. (b) Conditions as in (a) except that 2 pmol PE/PC, 70 pg bacteriorhodopsin, and 51 pg of mitochondrial F. were used. After 10 min incubation, 20-~1 samples were removed and added to 40 ~1 containing 250 mM sucrose, 50 mM Na-Tricine (pH 8.0), 0.5 mb! EDTA, 15 pg of F,, and 20 pg of OSCP, and incubated for 15 min at 22°C. The samples were assayed for ATP synthesis in one ml of reaction mixture.
RECONSTITUTION
OF MEMBRANE-BOUND
ENZYMES
475
Electron Microscopy of Reconstituted Cytochrome Oxidase Vesicles
% OCTYLGLUCOSIDE
FIG. 4. Reconstitution of Ca2+-ATPase from sarcoplasmic reticulum. In a final volume of 0.04 ml, 600 I*g sonicated asolectin in 0.2 M oxalate and the indicated concentration of octylglucoside were incubated at 0°C in the presence of 15 wg Ca2+-ATPase. After 15 s, 0.96 ml of a medium containing 50 mM Tris-HCI (pH ‘7.4), 100 mM KCl, 5 mM MgCl,, 0.1 mM CaCl, (0.15 &i Wa2+), 5 mM Tris-ATP (pH 6.8) was added. After 2 min incubation at 22°C an aliquot (0.90 ml) was withdrawn for Ca2+-transport assay. Assays performed in the absence of ATP served as controls.
unpublished experiments). In general the octylglucoside dilution procedure is more reproducible than methods involving sonication, particularly for brief time periods.
% OCTYLGLUCOSIDE
FIG. 5. Reconstitution of NaK-ATPase from electric eel. In a final volume of 0.12 ml, 3.19 pmol sonicated asolectin in 50 mM imidazole-H,SO1 (pH 7.51, ‘75 mM K,SOa, 50 mM Na,SO+ and 20 mM 2-mercaptoethanol and the indicated concentrations of octylglucoside were incubated at 0°C in the presence of 80 pg Na+K+ATPase. After 15 s, 3.0 ml of the above ice-cold buffer was added, the tubes gently vortexed, then centrifuged at 48,000 rpm for 30 min in a 50 Ti Beckman centrifuge rotor. Pellets were resuspended in 0.15 ml buffer and a 50+1 aliquot was withdrawn for Na+-transport assay for 2 min at 37°C. Assays were performed by the Dowex column procedure (19) and samples in the absence of ATP served as controls.
The various reconstitution procedures yield vesicles with different functional as well as morphological properties. The electron micrographs in Fig. 6 show that the sonication procedure yields smooth vesicles of various sizes, mostly small (about 500 A in diameter). The largest vesicles were obtained by octylglucoside dilution, many of them 2000 A or larger. Both freeze-thaw sonication and solvent sonication yielded rather irregular vesicles with a tendency to form aggregates. DISCUSSION
Of the various reconstitution procedures described thus far, the cholate dialysis procedure (6) has been the most widely used. The sonication procedure (7), particularly when preceeded by freezing and thawing (8), is more rapid but not as reproducible because of variability in sonicator energy output. The cholate dilution procedure (9) was found to be more reproducible, but was not as widely applicable, e.g., it failed with reconstitution of the bacteriorhodopsin proton pump. The detergent dilution procedure with octylglucoside described here is the most rapid, requiring only mixing of liposomes with the proteins in the presence of the detergent, followed by assay after appropriate dilution. However, failures with this method have been encountered. For example, in the case of acetylcholine receptor, octylglucoside was inhibitory, whereas cholate at appropriate concentrations was well tolerated (M. Schell and E. Racker, unpublished observations). We have therefore described variants of the dilution procedure in which other detergents and mixtures of ionic and nonionic detergents were used. One difficulty encountered with the dilution procedures is the requirement of a highly sensitive assay permitting 20- to 50-fold dilution of the reconstituted vesicles. This difficulty can be overcome by first diluting the reconstituted vesicles followed by centrifugation as described under Materials and Methods. For example, this procedure
RACKER
ET AL.
FIG. 6. Electron micrographs of reconstituted cytochrome oxidase vesicles. A copper grid (400 mesh) supporting a thin film of carbon was floated, briefly, carbon side down on a drop of the suspension of cytochrome oxidase vesicles. The grid was then placed, briefly, sample side down, on a drop of2% Naphosphotungstate (pH 7), the excess stain removed with filter paper, and the grid dried in a stream of Freon 12 gas. The samples were examined in a AEI EMGB electron microscope operated at an accelerating voltage of 80 kV and an instrument magnification of 20,000 X. Cytochrome oxidase vesicles were reconstituted by: (A) sonication (6 min); (B) freeze-thaw sonication (1 min); (Cl octylglucoside dilution; (D) solvent sonication (I-bromohexane). Magnification x59,000.
was used for the reconstitution of the Na+K+ATPase of plasma membranes. A second difficulty is that the optimal detergent concentration for the reconstitu-
tion of one protein may differ considerably for a second protein, thus rendering simultaneous reconstitution ineffective. In this case, sequential reconstitution (5) is ap-
RECONSTITUTION
OF MEMBRANE-BOUND
3. propriate. For example, in the case of the bacteriorhodopsin-chloroplast ATPase re4. constitution, bacteriorhodopsin vesicles are first formed at 1.25% octylglucoside, diluted 20-fold in 50 mM KCl, 10 mM Tricine (pH ELO), 5. and then centrifuged 10 min at 40,000 rpm. The resuspended vesicles are then reconstituted with the chloroplast ATPase at 0.8% 6. octylglucoside concentration. Attempts to use other nonionic detergents 7. instead of octylglucoside have thus far not 8. been encouraging. However, the remarkable variations of response to detergents by dif9. ferent membrane proteins on the one hand and the failure of octylglucoside to yield 10. active vesicles on the other, suggest that further explorations of detergents and detergent mixtures is called for. 11.
ACKNOWLEDGMENTS
12.
This work was supported by Grants CA-08964 and CA-14454, Grant BMS-7517337 from the National Science Foundation, awarded by the National Cancer Institute, DHEW, and National Institutes of Health Fellowships F-32GMO6467 (B.N.V.) and F-32CAO6053 (S.G.O.). M.A. has been supported by CDCH, Universidad Central de Venezuela, Caracas, Venezuela.
2.
Biochem. HORECKER,
F., AND WILLIAMSON, J. 64, 567-578.
14. 15. 16. 17.
REFERENCES 1. DICKENS,
13.
D.
H.
(1956)
B. L. (1951) in Phosphorus Metabolism (McElroy, W. D., and Glass, B., eds.), pp. 117144, The Johns Hopkins Press, Baltimore.
18.
19.
ENZYMES
477
DE LA HABA, G., LEDER, I., AND RACKER, E. (1955) J. Biol. Chem. 214, 409-426. SRERE, P. A., COOPER, J. R., KLYBAS, V., AND RACKER, E. (1955) Arch. Biochem. Biophys. 59, 535-538. RACKER, E. (1979) in Methods in Enzymology
(Fleischer, S., and Packer, L., eds.), Academic Press, New York, Vol. LV, pp. 699-711. KAGAWA, Y., AND RACKER, E. (1971) J. Biol. Chem. 246, 5477-5487. RACKER, E. (1973) Biochem. Biophys. Res. Commun. 55, 224-230. KASAHARA, M., AND HINKLE, P. C. (1976) Proc. Nat. Acad. Sci. USA 73, 396-400. RACKER, E., CHIEN, T-F., AND KANDRACH, A. (1975) FEBS Lett. 57, 14-18. BANERJEE, R., EPSTEIN, M., KANDRACH, M., ZIMNIAK, P., AND RACKER, E. (1979) Membrane Biochem., in press. RAGAN, C. I., AND RACKER, E. (1973) J. Biol. Chem. 248, 6876-6884. KANNER, B. I., AND RACKER, E. (1975) Biochem. Biophys. Res. Commun. 64, 1054-1061. CARROLL, R. C., AND RACKER, E. (1977) J. Biol. Chem. 252, 6981-6990. KURIKI, Y., AND RACKER, E. (1976) Biochemistry 15, 4951-4956. ALFONZO, M., AND RACKER, E. (1979) Fed. Proc. 38, 455. PICK, U., AND RACKER, E. (1979) J. Biol. Chem. 254, 2793-2799. RACKER, E., AND STOECKENIIJS, W. (1974) J. Biol. Chem. 249, 662-663. ZIMNIAK, P., AND RACKER, E. (1978) J. Biol. Chem. 253, 4631-4637. GASKO, O., KNOWLES, A., SHERTZER, H., SUOLINNA, E-M., ANDRACKER, E. (1976)Anal. Biochem. 72, 57-65.
Reconstitution,
a Way of Biochemical Research; Some New Approaches to Membrane-Bound Enzyme&
E. RACKER, Section
B. VIOLAND,
of Biochemistry,
Molecular
S. O’NEAL, & Cell
Biology,
M. ALFONZO, Cornell
University,
AND J. TELFORD Ithaca,
New
York
l&953
Received July 30, 1979 A variety of reconstitution procedures has been developed for the analysis of the biological activity of purified membrane proteins. In this paper we describe some new procedures. One is based on the action of two detergents, one ionic and one nonionic. Another method takes advantage of rather unique properties of octylglucoside which, in contrast to the detergents used in the first procedure, allows for virtually instantaneous reconstitution. This procedure is now the method of choice for the reconstitution of bacteriorhodopsin, and the proton pumps of mitochondria or chloroplasts. A third procedure takes advantage of the observation that very short periods of sonication are required when certain solvents (e.g., decane) are present in low concentrations.
In the course of studies on the hexose monophosphate shunt (the Warburg-Dickens pathway), two experiments were performed which illustrate the value of the resolution-reconstitution approach. In crude enzyme preparations from yeast, phosphogluconate was oxidatively decarboxylated with ribose 5-phosphate as a product of the reaction (1). However, with a purified enzyme ribulose 5-phosphate was formed and a second enzyme, an isomerase, was needed for the conversion to ribose li-phosphate (2). An enzyme from yeast which was crystallized and named transketolase (3) appeared to degrade ribulose 5-phosphate to glyceraldehyde 3-phosphate. However, after five to six recrystallizations, transketolase no longer degraded ribulose 5-phosphate and a second enzyme, an epimerase, had to be added to generate xylulose 5-phosphate, the substrate of transketolase (4). When we started to work on oxidative phosphorylation 20 years ago, we were naive enough to expect success with the same approach of resolution and reconstitution. It took about 10 years of frustration before we learned that this approach is 1 Dedicated to Professor Frank Dickens on the occasion of his eightieth birthday. 0003-9861/79/140470-08$02.00/O
Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
470
actually feasible. However, instead of allowing the reactions to take place in solutions as in the case of free-living enzymes, the membrane-bound enzymes had to be provided with special housing facilities. We had to build compartments for them in the form of phospholipid vesicles, called liposomes, which allowed the hydrophobic enzymes to assemble asymmetrically and fulfill their function as ion translocators. During the past 8 years we have developed a variety of methods of reconstitution into liposomes (5). The first, a cholate dialysis procedure (6) is still most widely used for a variety of membranous proteins. But some proteins do not tolerate exposure to cholate for prolonged periods and a sonication procedure was developed which avoided the use of detergents (7). Prolonged exposure to sonication is detrimental for some proteins and a new procedure, freeze-thaw sonication, was developed (8) which minimized the sonication time. A cholate dilution procedure (9) proved to be milder and more rapid than cholate dialysis. However, it is not applicable to some systems, e.g., bacteriorhodopsin, and often requires several hours of incubation. A survey of several other detergents and mixtures of detergents yielded suitable alternative methods which will be described
RECONSTITUTION TABLE EFFECT
OF MEMBRANE-BOUND
I
OF TIME OF INTERACTION BETWEEN CYTOCHROME OXIDASE AND PHOSPHOLIPIDS IN THE PRESENCE OF VARIOUS DETERGENT MIXTURES” Cholate (0.5%)octylglucoside (0.8%)
Cholate (1%) Time of incubation at 0°C
Respiratory control ratio
0 30 min 120 min 3h 20 h
1.0 1.2 1.3 1.7 4.0
Time of Respiratory incubation control at 0°C (min) ratio 0 15 30 90 160
1.0 2.5 3.5 4.5 5.0
a The experimental procedure was described under Materials and Methods. Crude soybean phospholipids were used at a lipid to protein ratio of 25. Cytochrome oxidase was present at a concentration of 1 mg/ml.
here. Particularly, octylglucoside proved most valuable for the reconstitution of bacteriorhodopsin and the proton pumps of mitochondria and chloroplasts. A new sonication procedure in the presence of low concentrations of solvents is also described. MATERIALS
AND METHODS
Preparations Cholic acid was recrystallized as described (6). Octylglucoside was either synthesized (10) or purchased from Calbiochem. Soybean phospholipids (asolectin) were obtained Tom Associated Concentrates, Woodside, New York. Phosphatidylethanolamine and phosphatidylcholine were prepared from bovine heart mitochondria (11). Bacteriorhodopsin (12), cytochrome oxidase (13), Ca*+-ATPase (lo), Na+K+-ATPase (14), and the F, complex of mitochondrial F,F,-ATPase2 (15) were purified as described in the references. Chloroplast ATPase complex was purified as described (16) except that lettuce leaves were used as the source and the extraction was performed with 0.44% octylglucoside ’ Abbreviations used: F,, couplingfactor 1 (ATPase); Fe, a membranous preparation from mitochondria conferring oligomycin (or rutamycin) sensitivity to F,; OSCP, oligomycin sensitivity conferring protein; PC, phosphatidylcholine; PE, phosphatidylethanolamine; Tricine, N-tris(hydroxymethyl)methylglycine.
ENZYMES
471
and 0.1% sodium cholate instead of the higher concentrations used with spinach leaves.
Reconstitution
Procedures
(a) Detmpnt &&ion. Phospholipids were sonicated in a bath-type sonic&or (7) to clarity (10 to 20 min) usually at 40 to 50 mM concentrations. They were mixed with protein, in the presence of detergent, at a phospholipid to protein ratio specified in the legends of tables and figures. After given time periods, samples were taken and diluted at least 20-fold in the enzyme assay mixture. If the sensitivity of the assay did not permit such a dilution, the samples were first diluted with buffer, centrifuged for 30 min at 55,000 rpm (65 Ti rotor in a Spinco centrifuge), suspended in a small volume of buffer, and then assayed. (b) Solvent sonication. Phospholipid suspensions were prepared and mixed with proteins as described under (a). Then a suitable amount of solvent (e.g., 2% 1-bromohexane) was added and the mixture was sonicated 15 to 30 s. The milky appearance of the suspension is caused by the solvent and can be observed in the absence of any phospholipids.
Assay Procedures Respiratory control of cytochrome oxidase (13), proton pumping by bacteriorhodopsin (17), 32P,-ATP exchange (16), Cal+ pump activity (18), and light-driven ATP formation (‘7) were assayed as described in the references. RESULTS
Reconstitution of Cytochrome Oxidase Vesicles with Respiratory Control Respiratory control developed slowly when cytochrome oxidase was incubated at 0°C with soybean phospholipids in the presence of 1% cholate. As shown in Table I, addition of octylglucoside greatly accelerated the reconstitution process. Optimal values were obtained when the concentrations of cholate and octylglucoside were carefully balanced against each other (Figs. 1A and B). The phospholipid to protein ratio in the experiment shown in Table I was 25 (w/w). With some preparations of cytochrome oxidase which were unstable under these conditions, a higher phospholipid to protein ratio was required (Table II). Octylglucoside can be used as the only detergent added at a 1.5% concentration (Table II). This is the most rapid reconstitution procedure ,since respiratory control is established with only a brief incubation of
472
RACKER
ET AL.
1
FIG. 1. (a) Titration of octylglucoside for the reconstitution of cytochrome oxidase vesicles with respiratory control. Asolectin lipsomes at 50 mM were prepared by sonication for 15 min in 50 mM IG', (pH 7.5) as described previously (6). Reconstitutions were performed for 2 h at 0°C in a medium containing 1 mg cytochrome oxidase/ml in the presence of 0.5% sodium cholate and varying amounts of octylglucoside as indicated. The phospholipid to protein ratio was 16:l. Ten microliters was used for each assay. (b) Titration of cholate for the reconstitution of cytochrome oxidase vesicles with respiratory control. The experimental conditions were as described for (a) with 0.7% octylglucoside and varying amounts of cholate as indicated.
the mixture before dilution. As will be apparent from subsequent data, each system has its own optimal condition for reconstitution. In the case of cytochrome oxidase the optimum octylglucoside concentration was close to 1.25% but respiratory control dropped sharply at slightly lower concentrations. A new procedure for the reconstitution of cytochrome oxidase vesicles is described in Table III. The method is a variant of the sonication method based on the observation that the sonication time can be drastically reduced if the liposome-protein mixture is sonicated in the presence of a solvent. A large number of various solvents were
tested and a representative group is listed in the table. Respiratory control ratios over 5 are readily obtained by this method. Reconstitution of the Bacteriorhodopsin Proton Pump Bacteriorhodopsin was readily incorporated into liposomes in the presence of 1.25 to 1.75% octylglucoside. Optimal proton pump activity upon dilution was achieved soon after mixing of the protein and phospholipid. Steady-state proton pumping in the light as well as initial rates of H+ translocation were high (4000 to 5000 n atom H+/ min/mg protein). These rates are consider-
TABLE
II
COMPARISONOFCHOLATE-OCTYLGLUCOSIDEANDOCTYLGLUCOSIDE DILUTION RECONSTITUTION OFCYTOCHROME OXIDASE VESICLES WITHRESPIRATORYCONTROL~ Cholate (0.7%)-octylglucoside
Experiment Experiment
1 2
(0.7%)
Time of incubation at 0°C (min)
Respiratory control ratio
60 60
2.2 4.1
Octylglucoside (1.5%) Time of incubation at 0” (min) 10 10
Respiratory control ratio 1.9 4.6
a The experimental procedure was as described in Table I, except that a different cytochrome oxidase preparation was used and the phospholipid to protein ratio was 25 in Experiment 1 and 40 in Experiment 2.
RECONSTITUTION TABLE
OF IVIEMBRANE-BOUND
III
SOLVENT SONICATION RECONSTITUTION OF CYTOCHROME OXIDASE~
Solvent
Ccl
Time of sonication (set)
1-Bromohexane 1-Bromohexane 1-Bromohexane 1-Bromohexane 1-Bromohexane I-Bromohexane 1-Bromohexane Tetradecane Decane Octane Heptane Hexane Pentane Pentane Toluene Ether Ether
1 1 2 2 3 4 5 4 4
15 30 15 30 15 15 15 15 15
4 4 8 2 2 2
15 15 30 30 15 30
Respiratory control ratio 1.6 2.1 2.3 6.0 3.3 3.8 4.0 2.3 4.9 4.2 4.6 3.3 1.5 1.9 2.2 2.2 2.0
ably higher than in liposomes reconstituted by other procedures such as cholate dialysis (17), direct sonication (7) or freeze-thaw sonication (8). A value for vesicles, obtained by the last procedure under experimental conditions comparable to those used for the octylglucoside dilution method, is shown in Table IV for comparison. Below 1% octylglucoside the reconstitution became ineffective. However, when cholate, which by itself did not facilitate reconstitution, was added to 1% octylglucoside, reconstitution took place. Once again, reconstitution was dependent on the incubation time of the detergent mixture. At 1% octylglucoside-0.25% cholate, it took 5 h incubation at 0°C to attain maximal H+ pumping rate. Reconstitution Chloroplasts Exchange
a In a final volume of 0.1 ml 4 pmol of sonicated asolectin (in the presence of 50 mM KP,, pH 7.5) were mixed in a small test tube (12 x 74 mm) with 120 pg of cytochrome oxidase and 2 to 4 ~1 of solvent. The suspension appeared milky after brief sonication in a water bath sonicator. Respiratory control ratio was determined by measuring oxygen uptake in the absence and presence of valinomycin (2 pg/ml)-nigericin
(2 j@nl). TABLE
473
ENZYMES
of the H+-ATPase from Catalyzing 32Pi-ATP
The best reconstitution of the chloroplast ATPase complex described thus far is the freeze-thaw procedure with no sonication (16). The octylglucoside dilution procedure gave even higher activity and was very reproducible. It is apparent from Fig. 2 that the concentration of octylglucoside required for optimal reconstitution of the 32Pi-ATP exchange varied with the concentration of phospholipids and protein. IV
RECONSTITUTION OF BACTERIORHODOPSIN WITH OCTYLGLUCOSIDE OR OCTYLGLUCOSIDE-CHOLATE MIXTURES’ Experiment Experiment
2
1
Concentration of octylglucoside (%)
n atoms H+/ mg protein
Concentration of cholate (with 1% octylglucoside) (%)
0.5 1.0 1.25 1.50 1.75 2.0 4.2
0 120 702 601 538 394 212
0 0.125 0.250 0.370 0.250 (no octylglucoside) Liposomes prepared by freeze-thaw sonication
n atoms H+/ mg protein 135 725 705 411 0 173
a In a final volume of 40 ~1 containing 75 mM KCl, 1 mM Na-Tricine (pH 8.0), 0.8 pmol of sonicated asolectin was mixed with 10 pg of bacteriorhodopsin in the presence of the indicated concentrations of detergent. The entire sample was added to 1 ml of 0.15 M KC1 for assay.
474
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ET AL.
Reconstitution of Purified Mitochondrial ATPase Complex and Bacteriorhodopsin Catal yxing ATP Formation As shown in Fig. 3, simultaneous reconstitution of two different functional proteins by the octylglucoside dilution procedure is possible if there is enough overlap with respect to reconstitution conditions for each component. The rates of ATP formation in the light in this experiment were two- to fivefold higher than previously reported for either the cholate dialysis (17) or the sonication (7) procedures. Freeze-thaw sonication (8) also gave lower activity than octylglucoside dilution (Fig. 3). The need for careful titration of octylglucoside with different protein and phospholipid concentrations is illustrated in Fig. 3. It can be seen that the optimal concentration for one mixture may be completely ineffective for another. Since lower concentrations were needed for the chloroplast ATPase than for bacteriorhodopsin, simultaneous incorporation did not yield very active preparations. In such a case sequential reconstitution (5) can be used as will be described below. Reconstitution of Sarcoplasmic Reticulum Ca2+-ATPase Catalyzing Ca2+ Transport As shown in Fig. 4, the purified Ca2+ATPase from sarcoplasmic reticulum was incorporated into liposomes at various concentrations of octylglucoside. It should be noted that 1.2596, the optimal concentration for reconstitution of bacteriorhodopsin (Table III), is virtually ineffective for the Ca*+-ATPase. Experiments similar to those shown in Fig. 4 were performed with sarcoplasmic reticulum instead of purified enzyme with comparable results. Reconstitution Catalyzing
of Na+K+-ATPase Na+ Uptake
As shown in Fig. 5, purified Na+K+ATPase from electric eel was incorporated into liposomes at various concentrations of octylglucoside. It can be seen that higher concentrations of octylglucoside are required compared to reconstitutions of other membrane proteins, but in general the titration is similar to that observed with the Ca*+-
FIG. 2. Reconstitution of chloroplast H+-ATPase catalyzing 32P,-ATP exchange activity. (a) In a final volume of 0.1 ml containing 3.5 mg sonicated asolectin, 80 mM Na-Tricine (pH 8.0), 0.1 mM EDTA, and oetylglucoside as indicated were incubated at 0°C in the presence of 100 pg chloroplast ATPase complex. After 10 min, lo-p1 samples were assayed for 3ZPi-ATP exchange in l-ml reaction mixture. (b) Conditions were as in (a) except that 2 mg sonicated asolectin and 50 pg of the ATPase complex were used.
ATPase (Fig. 4). The ratio of Na+ transport is high compared to that obtained by- the freeze-thaw sonication procedure (D. Cohn,
1 FIG. 3. Reconstitution of mitoehondrial H+-ATPase complex and bacteriorhodopsin catalyzing ATP formation. (a) In a final volume of 0.1 ml, 0.625 pmol sonicated PE/PC (1:l molar ratio), 10 mM Na-Tricine (pH 8.0), 10 mM KCl, and octylglucoside as indicated were incubated at 0°C in the presence of 80 pg bacteriorhodopsin and 32 pg of mitochondrial F,. (b) Conditions as in (a) except that 2 pmol PE/PC, 70 pg bacteriorhodopsin, and 51 pg of mitochondrial F. were used. After 10 min incubation, 20-~1 samples were removed and added to 40 ~1 containing 250 mM sucrose, 50 mM Na-Tricine (pH 8.0), 0.5 mb! EDTA, 15 pg of F,, and 20 pg of OSCP, and incubated for 15 min at 22°C. The samples were assayed for ATP synthesis in one ml of reaction mixture.
RECONSTITUTION
OF MEMBRANE-BOUND
ENZYMES
475
Electron Microscopy of Reconstituted Cytochrome Oxidase Vesicles
% OCTYLGLUCOSIDE
FIG. 4. Reconstitution of Ca2+-ATPase from sarcoplasmic reticulum. In a final volume of 0.04 ml, 600 I*g sonicated asolectin in 0.2 M oxalate and the indicated concentration of octylglucoside were incubated at 0°C in the presence of 15 wg Ca2+-ATPase. After 15 s, 0.96 ml of a medium containing 50 mM Tris-HCI (pH ‘7.4), 100 mM KCl, 5 mM MgCl,, 0.1 mM CaCl, (0.15 &i Wa2+), 5 mM Tris-ATP (pH 6.8) was added. After 2 min incubation at 22°C an aliquot (0.90 ml) was withdrawn for Ca2+-transport assay. Assays performed in the absence of ATP served as controls.
unpublished experiments). In general the octylglucoside dilution procedure is more reproducible than methods involving sonication, particularly for brief time periods.
% OCTYLGLUCOSIDE
FIG. 5. Reconstitution of NaK-ATPase from electric eel. In a final volume of 0.12 ml, 3.19 pmol sonicated asolectin in 50 mM imidazole-H,SO1 (pH 7.51, ‘75 mM K,SOa, 50 mM Na,SO+ and 20 mM 2-mercaptoethanol and the indicated concentrations of octylglucoside were incubated at 0°C in the presence of 80 pg Na+K+ATPase. After 15 s, 3.0 ml of the above ice-cold buffer was added, the tubes gently vortexed, then centrifuged at 48,000 rpm for 30 min in a 50 Ti Beckman centrifuge rotor. Pellets were resuspended in 0.15 ml buffer and a 50+1 aliquot was withdrawn for Na+-transport assay for 2 min at 37°C. Assays were performed by the Dowex column procedure (19) and samples in the absence of ATP served as controls.
The various reconstitution procedures yield vesicles with different functional as well as morphological properties. The electron micrographs in Fig. 6 show that the sonication procedure yields smooth vesicles of various sizes, mostly small (about 500 A in diameter). The largest vesicles were obtained by octylglucoside dilution, many of them 2000 A or larger. Both freeze-thaw sonication and solvent sonication yielded rather irregular vesicles with a tendency to form aggregates. DISCUSSION
Of the various reconstitution procedures described thus far, the cholate dialysis procedure (6) has been the most widely used. The sonication procedure (7), particularly when preceeded by freezing and thawing (8), is more rapid but not as reproducible because of variability in sonicator energy output. The cholate dilution procedure (9) was found to be more reproducible, but was not as widely applicable, e.g., it failed with reconstitution of the bacteriorhodopsin proton pump. The detergent dilution procedure with octylglucoside described here is the most rapid, requiring only mixing of liposomes with the proteins in the presence of the detergent, followed by assay after appropriate dilution. However, failures with this method have been encountered. For example, in the case of acetylcholine receptor, octylglucoside was inhibitory, whereas cholate at appropriate concentrations was well tolerated (M. Schell and E. Racker, unpublished observations). We have therefore described variants of the dilution procedure in which other detergents and mixtures of ionic and nonionic detergents were used. One difficulty encountered with the dilution procedures is the requirement of a highly sensitive assay permitting 20- to 50-fold dilution of the reconstituted vesicles. This difficulty can be overcome by first diluting the reconstituted vesicles followed by centrifugation as described under Materials and Methods. For example, this procedure
RACKER
ET AL.
FIG. 6. Electron micrographs of reconstituted cytochrome oxidase vesicles. A copper grid (400 mesh) supporting a thin film of carbon was floated, briefly, carbon side down on a drop of the suspension of cytochrome oxidase vesicles. The grid was then placed, briefly, sample side down, on a drop of2% Naphosphotungstate (pH 7), the excess stain removed with filter paper, and the grid dried in a stream of Freon 12 gas. The samples were examined in a AEI EMGB electron microscope operated at an accelerating voltage of 80 kV and an instrument magnification of 20,000 X. Cytochrome oxidase vesicles were reconstituted by: (A) sonication (6 min); (B) freeze-thaw sonication (1 min); (Cl octylglucoside dilution; (D) solvent sonication (I-bromohexane). Magnification x59,000.
was used for the reconstitution of the Na+K+ATPase of plasma membranes. A second difficulty is that the optimal detergent concentration for the reconstitu-
tion of one protein may differ considerably for a second protein, thus rendering simultaneous reconstitution ineffective. In this case, sequential reconstitution (5) is ap-
RECONSTITUTION
OF MEMBRANE-BOUND
3. propriate. For example, in the case of the bacteriorhodopsin-chloroplast ATPase re4. constitution, bacteriorhodopsin vesicles are first formed at 1.25% octylglucoside, diluted 20-fold in 50 mM KCl, 10 mM Tricine (pH ELO), 5. and then centrifuged 10 min at 40,000 rpm. The resuspended vesicles are then reconstituted with the chloroplast ATPase at 0.8% 6. octylglucoside concentration. Attempts to use other nonionic detergents 7. instead of octylglucoside have thus far not 8. been encouraging. However, the remarkable variations of response to detergents by dif9. ferent membrane proteins on the one hand and the failure of octylglucoside to yield 10. active vesicles on the other, suggest that further explorations of detergents and detergent mixtures is called for. 11.
ACKNOWLEDGMENTS
12.
This work was supported by Grants CA-08964 and CA-14454, Grant BMS-7517337 from the National Science Foundation, awarded by the National Cancer Institute, DHEW, and National Institutes of Health Fellowships F-32GMO6467 (B.N.V.) and F-32CAO6053 (S.G.O.). M.A. has been supported by CDCH, Universidad Central de Venezuela, Caracas, Venezuela.
2.
Biochem. HORECKER,
F., AND WILLIAMSON, J. 64, 567-578.
14. 15. 16. 17.
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19.
ENZYMES
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DE LA HABA, G., LEDER, I., AND RACKER, E. (1955) J. Biol. Chem. 214, 409-426. SRERE, P. A., COOPER, J. R., KLYBAS, V., AND RACKER, E. (1955) Arch. Biochem. Biophys. 59, 535-538. RACKER, E. (1979) in Methods in Enzymology
(Fleischer, S., and Packer, L., eds.), Academic Press, New York, Vol. LV, pp. 699-711. KAGAWA, Y., AND RACKER, E. (1971) J. Biol. Chem. 246, 5477-5487. RACKER, E. (1973) Biochem. Biophys. Res. Commun. 55, 224-230. KASAHARA, M., AND HINKLE, P. C. (1976) Proc. Nat. Acad. Sci. USA 73, 396-400. RACKER, E., CHIEN, T-F., AND KANDRACH, A. (1975) FEBS Lett. 57, 14-18. BANERJEE, R., EPSTEIN, M., KANDRACH, M., ZIMNIAK, P., AND RACKER, E. (1979) Membrane Biochem., in press. RAGAN, C. I., AND RACKER, E. (1973) J. Biol. Chem. 248, 6876-6884. KANNER, B. I., AND RACKER, E. (1975) Biochem. Biophys. Res. Commun. 64, 1054-1061. CARROLL, R. C., AND RACKER, E. (1977) J. Biol. Chem. 252, 6981-6990. KURIKI, Y., AND RACKER, E. (1976) Biochemistry 15, 4951-4956. ALFONZO, M., AND RACKER, E. (1979) Fed. Proc. 38, 455. PICK, U., AND RACKER, E. (1979) J. Biol. Chem. 254, 2793-2799. RACKER, E., AND STOECKENIIJS, W. (1974) J. Biol. Chem. 249, 662-663. ZIMNIAK, P., AND RACKER, E. (1978) J. Biol. Chem. 253, 4631-4637. GASKO, O., KNOWLES, A., SHERTZER, H., SUOLINNA, E-M., ANDRACKER, E. (1976)Anal. Biochem. 72, 57-65.