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Use of the Airfuge for analysis and preparation of receptors incorporated into liposomes: Studies with the receptor for immunoglobulin E
ANALYTICAL
BIOCHEMISTRY
130,
514-520
(1983)
Use of the Airfuge for Analysis and Preparation of Receptors Incorporated into Liposomes: Studies with the Receptor for lmmunoglobulin E BENJAMIN RIVNAY’ AND HENRY METZGER~ Section on Chemical Immunology, Arthritis and Rheumatism Branch, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205 Received September 23, 1982 A Beckman Airfuge has been employed for studying the interaction between lipids and the receptor for immunoglobulin E (IgE). For analytic experiments, samples were applied underneath a discontinuous sucrose gradient. After a 30-min centrifugation in a fixed-angle rotor, liposomes floated toward the top of the gradient whereas unincorporated receptor-IgE complexes remained at the bottom of the tube. Liposomes with incorporated receptors were also efficiently separated in the ACR-90 preparative rotor. These methods of “Airfuge flotation” can provide useful adjuncts to more traditional methods for density-gradient centrifugation especially when rapid analysis of small samples is desired.
In most work on the reconstitution of membrane proteins, a mixture of lipids, protein, and detergent is dialyzed to remove the detergent. Whereas the protein is often efficiently incorporated into the liposomes formed in this way, this is not always so. It therefore becomes necessary to analyze the dialyzed mixture or to separate the unincorporated material for preparative purposes. This proved to be the case in our investigations on the receptor for immunoglobulin E (IgE)3 (1). In those experiments we analyzed the mixtures by the traditional procedure of sucrose-density-gradient centrifugation in swinging-bucket rotors utilizing an ultracentrifuge. During the course of those studies we explored the possibility of using much shorter centrifugations in the Beckman Airfuge as an adjunct or alternative to such analyses. In this paper we describe the procedures which we found to be useful. ’ Present address: Weizmann Institute of Science, Rehovot, Israel. 2 To whom correspondence should be addressed at: Building 10, Room 9N-2 18, National Institutes of Health, Bethesda, Md. 20205. r Abbreviation used: IgE, immunoglobuhn E. 0003-2697183
$3.00
Copyright Q 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
514
MATERIALS
AND METHODS
Chemicals. Rat immunoglobulin E was isolated from as&tic fluid of tumor IR 162 as described by Isersky et al. (2) and by Kulczycki and Metzger (3), and iodinated by the chloramine-T method (4, 5) using Na1251 (Amersham, Arlington Heights, Ill.). Cytochrome c from horse heart, Type III (No. C-2506), 1-cu-phosphatidylcholine from soybean (No. P-3644), 1-O-n-octyl-P-D©ranoside (octylglucoside, No. O-800 l), iodoacetamide (No. I-6 125), phenylmethylsulfonyl fluoride (No. P-7626), aprotinin from bovine lung (No. A-60 12) and pepstatin (No. P-4265) were purchased from Sigma (St. Louis, MO.). Leupeptin was brought from Boehringer-Mannheim Biochemicals (Indianapolis, Ind.) and sucrose (density gradient grade) from Schwartz/Mann (Orangeburg, N. Y.). The buffer used throughout this study was Tris-saline (15 mM Tris-HCl, pH 7.6, 5 mM KCl, and 125 mM NaCl). Liposomes. Sonicated liposomes were prepared by thoroughly drying a chloroform solution of bis( [ 1-14C]palmitoyl)phosphati-
AIRFUGE
FLOTATION
dylcholine and soybean lecithin, swelling in Tris buffer at a concentration of 50 mg/ml, and sonicating with the probe of a sonicator (Model W225R, Heat Systems, Plainview, N. Y.) at 70% output on ice. The supernatant was used after centrifugation at 3 1,OOOg,, at 4°C for 30 min. Liposomes prepared by the dialysis method were prepared in a similar way to that described in the section on Reconstitution except that the mixture did not contain cell extract or cytochrome c. Receptor solubilization. Rat basophilic leukemia cells (2H3 subline (6)), to which 1251IgE was bound, were solubilized with octylglucoside at final concentrations of 5 X 10’ cells/ml (25 nM in receptor-bound IgE) and 40 mM octylglucoside, respectively. Six inhibitors of proteolysis were added: 20 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 25 pg/ml aprotinin, 3 pg/ml pepstatin, and 1 pg/ml leupeptin. The extract was incubated 10 min on ice, centrifuged for 1 h at 3 1,OOOg,, at 4°C and the supernatant used immediately or stored at -80°C. The concentration of endogenous phospholipids was -2
rnM.
Reconstitution. Eighty microliters of detergent extract containing receptor-IgE complexes (cf. Receptor Solubilization) was mixed with exogenous lipids (soybean lecithin liposomes) and additional detergent to final concentrations of 5 nM bound IgE, 16 mM soybean lecithin, 200 mM octylglucoside, and 2 mg/ml cytochrome c (as carrier), and incubated on ice for 2 h. The detergent was removed by dialysis against 500 vol of Tris buffer with 3-4 daily changes for 3-4 days. Acid dissociation of IgE. IgE was dissociated from the reconstituted liposomes by acid treatment. Sixty-four microliters of the reconstituted liposomes in 15 mM Tris-HCl, pH 7.5, was incubated with 20 ~1 of 100 mM glycine . HCl, pH 2.35, (final pH 3) for 1 min, and then neutralized with 73 ~1 of a 150 mM Tris buffer (pH 8.1) containing 60% sucrose and 10 pg/ml unlabeled IgE (final pH 7.5). All incubations were done on ice. Gradient centrifugation. Stock solutions of 5, 10, 20,25,27, and 60% sucrose (w/w) were
OF
515
LIPOSOMES
prepared and checked by refractive index measurements on an Abbe-type refractometer (Bausch & Lomb, Rochester, N. Y .). Stepwise gradients of 4-5 layers were sequentially applied in Beckman Airfiuge test tubes (cellulose nitrate No. 339647 (4.8 X 19.9 mm) or Ultraclear No. 344718 (5 X 20 mm), Beckman, Palo Alto, Calif.) for a total gradient volume of 145- 150 ~1 (excluding the samplecontaining layer of 30-40 ~1). The compositions of gradients used in individual experiments are detailed in the legends to the figures. Since differences of 50-60 mg in tare weight were often found between individual test tubes, pairs of test tubes were weighed and matched within l-2 mg as a precaution to minimize rotor wobbling during acceleration and deceleration. In order to avoid turbulence and intermixing of the sucrose layers during construction of the gradient, the test tubes were frozen on dry ice for 1 min after each addition of a new layer. The work was facilitated by the use of a tube holder consisting of an aluminum block which was placed in granular dry ice. Twelve gradients of five steps were readily prepared in less than 15 min. The gradients were thawed and the specimen applied with a Hamilton syringe prior to the run. Centrifugation was carried out in a cold room (4°C) at top speed (30 psi) and operated from a wall outlet of pressurized air. Fractions of 25 ~1 were collected from the top with a pipettor held parallel to the tube, and with minimal insertion of the tip into the gradient. Counting. ‘251 counting was performed in a Beckman Gamma 8000 (Palo Alto, Calif.). 14Cradioactivity was counted in Aquasol (New England Nuclear, Boston, Mass.) in a Mark III scintillation counter (Tracer, Elk Grove Village, Ill.) with the 14C channel set to 65400 keV. RESULTS
Analytic separations. Density-gradient centrifugations are commonly performed in rotors with swinging buckets thereby permitting the gradient vector to remain parallel to the sides of the tube throughout the run. In a fixed angle rotor this relationship is not maintained
RIVNAY
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AND METZGER
thereby potentially causing destruction of the gradient. We tested to see if this would vitiate the possibility of using the fixed angle (0 = 18 “) rotor of the Airfuge. Figure 1 shows the results of two experiments employing duplicate discontinuous sucrose gradients which were centrifuged for 30 min (Fig. IA) and 40 min (Fig. lB), respectively. The patterns of the recovered gradients are superimposed over the applied gradients. As can be seen, the average slope of the recovered gradients was only about one-half of that of the gradient prior to centrifugation. However, it is apparent that there FRACTION
12345676 I
r
xl-
I
FRACTION
I
A
20-
2oLP-
i
FIG. 2. Liposome migration in Airfuge gradients. Four gradients containing 35 ~1 30%, 45 ~1 25%, 45 ~1 208, 30 ~1 IO%, and 20 pl 5% sucrose were prepared. In (A) the liposomes were contained in the top fraction. In (B) 20 ~1 of W-labeled liposomes in 30% sucrose were inserted as the lower part of the bottom fraction. One gradient in each set contained sonicated liposomes (0 - - - 0), the other one contained liposomes formed by detergent dialysis (0 0). Gradients were run and collected as detailed under Materials and Methods and counted in the scintillation counter. Vertical bars represent the radioactivity which remained in the empty test tubes: open bars, ?-lipids; filled bars, izsI-IgE.
was a monotonic increase in the density of the specimens recovered by taking successive L-J samples from the top of the tube. Moreover, the results from the duplicate tubes showed excellent reproducibility. No change in the VOLUME (pL1 slope of the recovered gradient was observed FIG. 1. Stability of stepwise gradients centrifuged in the when the tubes were left in a vertical position A- 100 (fixed angle) rotor in the Airfuge. (A) Two gradients containing 50 ~1 of 296, IO fll 25%, 30 ~1 20%, 45 ~1 for 1 h after centrifugation. The last fraction IO%, and 40 ~1 5% sucrose were. run for 30 min. 25-~1 usually had a slightly lower density than the fractions were collected from the top, and the refractive penultimate one (Fig. 1), probably due to slidindices measured and translated to sucrose concentrations. One gradient (-) was analyzed immediately, and ing of some of the top fraction down the sides the other (- - -) one hour later. (B) Two gradients (solid of the tube during removal of successive fracand dashed lines) with 35 ~1 of 30%, 40 pl 26%, 35 pi tions. We assessed whether it would be better 25%, 30 pl20%, 25 ~1 5% sucrose, and 20 ~1 Tris buffer to apply the samples on top of, or beneath, were run for 40 min. Fractions were immediately col- the gradient. Figure 2 shows the distribution lected from both gradients. (O 0) pattern of the original gradient. Arrows indicate the lower density of the of two types of liposomes when applied to the volume remaining for the partial 8th fraction. See also top or underneath identical gradients. Neither Fig. 4 for the choice of centrifugation period. the liposomes prepared by sonication (small, 10
AIRFUGE
FLOTATION
unilamellar) nor those prepared by detergent dialysis (large uni- and multilamellar) sedimented when applied to the top of the gradient (Fig. 2A). However, when the sample was applied under the gradient a differential flotation was observed (Fig. 2B). The liposomes formed by dialysis which have a lower density because they contain a large internal volume of sucrose-free solvent, floated to the top of the gradient whereas the sonicated vesicles floated only halfway up the gradient. A comparable experiment employing unbound IgE showed the expected result. When applied under the gradient, the IgE remained in the bottom fractions; when applied to the top of the gradient, it failed to sediment significantly during the 30-min centrifugation. The combined results with the liposomes and the IgE indicated that in short centrifugations the specimens had to be applied underneath the gradient in order to separate liposome-bound from unincorporated IgE-receptor complexes. We refer to this procedure as “Airfuge flotation.” After numerous experiments with patterns similar to that shown in Fig. 2B, we chose to consider the material in the top four fractions as representing liposome-associated components. Recoveries of 14C-lipids in these fractions routinely ranged between 65 and 85% of the applied amount; the remainder was found in the bottom fractions and in the empty test tube (~9%). Several examples of Airfuge flotation will now be given. In one experiment we wished to determine if the speed of removal of the detergent would affect the incorporation. We prepared a detergent extract of cells to which ‘251-IgE had been bound. The extract was supplemented with asolectin, octylglucoside, and cytochrome c (as carrier). One aliquot was thoroughly dialyzed (see Materials and Methods): the other was passed over a column of Sephadex G-25 (medium) and the fractions from the void volume were collected. Figure 3 depicts the analysis of the two separations. With the dialyzed sample -35% of the ‘251labeled IgE and -83% of the i4C-lipids were
517
OF LIPOSOMES
1
3
5
7
FRACTION
FIG. 3. Airfuge flotation of vesicles reconstituted with crude extracts of cells containing &-receptor complexes and soybean lecithin. The 1251-IgE-receptors were solubilked as detailed under Materials and Methods mixed with additional octylghtcoside (200 mM), soybean lecithin (16 mM), and with cytochrome c (2 mg/ml) as carrier, and incubated 2 h on ice. Two hundred microliters of the mixture was dialyzed thoroughly (A), and 90 ~1 was passed through a 1.5 X 13-cm column of Sephadex G-25 medium (B). The reconstituted material (from (A) and (B)) was made 30% in sucrose, and 35 ~1 floated in duplicate gradients composed of 45 ~125%, 40 ~120%, 25 ~1 IO%, and 30 ~1 5% sucrose. After 30 min centrifugation, the gradients were analyzed as described. Bars represent Wlipids (0) or ‘Z51-IgE (m) counts remaining in the empty test tubes. The combined gradients in panel (A) contained 11,700 cpm of 14Cand 8860 cpm of ‘251;and in (B), 7050 and 5 100, respectively.
recovered in the top four fractions of the gradient (Fig. 3A). With the sample filtered over Sephadex the comparable fractions contained -76% of the lipids but only - 19% of the IgE. If we assume that those lipids which were not recovered in the top four fractions nevertheless represented liposomal material, then the “corrected” values for the liposome-associated h&receptor complexes are 42 and 25%, respectively. On the basis of these results we adopted dialysis as the method of choice. In the initial phase of this study, a 30-min centrifugation was chosen arbitrarily. In order to check whether this time was appropriate, a preparation which contained approximately
RIVNAY
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AND METZGER
equal amounts of free and liposome-associated receptor-IgE complexes was centrifuged during 10, 20, or 40 min at 30 psi. Though flotation was somewhat better after 40 min than after 20 min (Fig. 4), the relative recoveries of the i4C-lipids and of the ‘251-IgE counts (not shown for sake of clarity) in the top four fractions were very similar. After 10 min of centrifugation the liposomes migrated only half way and the separation from the unincorporated complexes was poor. When two Airfuges are connected in tandem, thereby permitting 12 samples to be centrifuged simultaneously, the air pressure (obtained from a wall outlet) may be somewhat lower (e.g., 20-25 psi). This in turn reduces the speed of centrifugation - 10 percent. We checked the effect of this variable. Identical gradients were centrifuged for 40 min with an air pressure of 20 psi (instead of 30 psi). Under these conditions the peak of the liposomes was at fractions 2 and 3, still well separated from the unincorporated receptors. Preparative separations. We tested to see if preparative separations could be performed by using the ACR-90 rotor (Fig. 5A). This
G $40
U-JO 03 his ug :! 5
E
40 min. -20 min.
10 min.
20
r!iiiil 1
3
5
7
FRACTION
FIG. 4. Length ofcentrifugation and liposome flotation. IgE-receptor complexes were reconstituted by dialysis as described under Materials and Methods and analyzed on gradients containing 40 ~1 27%, 35 ~1 25%, 30 ~1 20%, 25 ~1 5% and 20 ~1 0% sucrose. Identical samples were made 30% in sucrose, immediately centrifuged for the indicated periods of time, and analyzed as described under Materials and Methods. ‘*‘I counts recovered in the top 4 fractions were 35, 36, and 21% at 40, 20, and 10 min, respectively. The corrected values were calculated to be 41, 42, and 35% in the respective gradients. Less than 0.5% of the i4C cpm were associated with the empty test tubes.
A
liner adapter
outer inner
chamber rotor
outer
1
ring
chamber
base
chamber
3
5 FRACTION
7
FIG. 5. Isolation of liposomes with the ACR-90 rotor. A specimen that has been reconstituted from an octylglucoside-solubilized extract of cells with soybean lecithin (see Materials and Methods) was made 30% in sucrose and floated in the small liner (2.4 ml) of the ACR-90 rotor. (A) Schematic cross section of the ACR-90 rotor. 1 ml of 16% sucrose was injected into the outer chamber (1.4 ml) of the liner, and 0.7 ml of the specimen was injected underneath, causing an overtlow of the lighter cushion into the inner chamber (- 1 ml). The volume occupied by the specimen is marked by the horizontal dotted line. The inner chamber was then filled with 16% sucrose, the lid closed, and the rotor run for 75 min. The contents of the inner chamber were removed and fractions of 300 ~1 were collected from the bottom of the outer chamber. All additions and withdrawals were performed with a I-ml syringe attached to a blunt 23G needle. Horizontal bar in top right comer = 1 cm. This Figure is modified from Beckman Manual No. AF-TBOOZD, Feb. 1979. (B) Distribution of radioactivity in the inner chamber and the four fractions of the outer chamber. (C) Airfuge flotation analysis of the unfractionated material performed with the A-100 rotor. (D) Analysis of a sample from the inner chamber performed as in (C). Open symbols or open bars, 14C-lipids; filled circles and bars, ‘*‘IIgE. Bars in panels (C) and (D) represent material remaining in the empty test tubes.
AIRFUGE
FLOTATION
rotor was designed for clarification of lipemic sera and separation of chylomicrons. A reconstitution mixture was dialyzed and made 30% in sucrose. It was applied in the bottom of the outer chamber of the rotor liner under a layer of 16% sucrose as detailed in the legend to Fig. 5. The rotor was accelerated slowly (46 min) in order to avoid intermixing of the two sucrose layers and was then spun for 75 min. The entire contents (- 800 ~1) from the inner chamber were withdrawn without removing the rotor from the Airfuge, and then four fractions were collected from the bottom of the outer chamber. Analysis of the specimens (Fig. 5B) showed that more than 90% of the 14C-lipids were recovered in the inner chamber and about 50% of the 1251-IgE was found in this pool. Airfuge flotation in the A100 analytic rotor was performed on the unseparated mixture (Fig. 5C) as well as on the material recovered from the inner chamber (Fig. 5D). It can be seen that the unfractionated material (Fig. 5C) showed a distribution of 1251-IgE-receptor complexes consistent with the results obtained in the preparative run; the corrected value for incorporation was 46%. On the other hand, the sample from which the unincorporated receptors had been removed in the preparative centrifugation (Fig. 5D) now shows a virtually perfect concordance between the distribution of radioactivity associated with the lipids and that associated with the IgE-receptor complexes. The percentage of receptor complexes associated with the liposomes was calculated to be 98%. Airfuge flotation was also used to characterize the incorporated IgE-receptor complexes in terms of exposure to the external medium. Exposure of cells to acid (pH 3) rapidly dissociates the bound IgE (3). We have measured the dissociable fraction of IgE on the reconstituted liposomes in order to assess the amount putatively exposed on the outer surface of the liposomes. Vesicles containing IgE-receptor complexes were preparatively separated from the unincorporated material as described for Fig. 5 and the liposomes from
519
OF LIPOSOMES
the inner chamber were recovered. One aliquot was treated with acid for 1 min, and then was neutralized with Tris buffer containing unlabeled IgE (Fig. 6B). A control aliquot was treated with the same solutions as above, but these had been premixed (Fig. 6A). Only 37% of the labeled IgE remained associated with the liposomes after the acid treatment whereas the control sample showed 9 1% association. This suggested that approximately two-thirds of the complexes were properly oriented. We noted that the liposomes became more turbid upon acidifying and remained so after neutralization. It is also apparent that the treated liposomes migrated differently than the shamtreated material in the gradients (of Figs. 6A and B). It is possible that the acid pH’s caused the liposomes to fuse with each other but we have not checked this. Parallel experiments, in which rebinding of IgE to acid-dissociated complexes was studied (data not shown), showed quantitative rebinding of new IgE to the vacant receptors. This excluded the possibility that the receptors themselves had been detached from the liposomes at the acid pH.
1
3
5
7
FRACTION
FIG. 6. Acid-induced dissociation of IgE from reconstituted liposomes. (A) Control: separated liposomes were incubated with premixed acid and neutralizing buffers (Materials and Methods), and then analyzed by Airfuge flotation. (B) Acid treatment: hposomes and buffers like in (A) were mixed sequentially, the neutralizing buffer was added 1 min after the acid buffer, and the mixture was then analyzed as in (A). 0 - - - 0, “C-lipids; 00 ‘251-IgE. Filled and empty bars represent the “‘1 and I& counts in the empty test tube, respectively.
RIVNAY
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AND METZGER
DISCUSSION
We have presented a rapid semiquantitative method for analyzing protein-liposome interactions. The method is based on the differential flotation of the constituents in the mixture on sucrose gradients, utilizing a commercially available small air-driven centrifuge (Airfuge). Both analytical runs of six specimens of ~50 ~1 and preparative separations of ~750 ~1 can be performed, utilizing the A100 and ACR-90 rotors, respectively. The procedure yields reproducible separations. Duplicate samples generally give distributions of the components within 5% of each other, when run on the same gradients in different centrifugations. The method is economic in both price and space, by the nature of the equipment employed. It also uses little material and is rapid. Many steps in the procedure take less time than the parallel ones in the full-size ultracentrifuge. The most important difference is the time of centrifugation. We routinely used 30 min per run, which can be compared to the 18- to 36-h runs required in our earlier work using standard methods. Thirty centrifugations (180 tubes) can, in theory, be analyzed during the period of one overnight run in the ultracentrifuge; in practice, 4-5 centrifugations (24-30 tubes) were analyzed in one day by one person.4 We have illustrated the usefulness of the method with two sets of experiments involving the reconstitution of the @-receptor complexes. In the first, we demonstrated that dialysis was preferable to gel filtration for removal of the detergent. Earlier reconstitution studies with other systems (7,8) indicated that,
with Sephadex filtration or rapid dilution, less or improper reconstitution occurred. Failure to achieve efficiencies higher than 40-50% (Figs. 3 and 5) is related, in the case of the IgE-receptor system, to the nature of the detergent (octylglucoside) and the lipid (asolectin) used (l), and no improvement was observed when the detergent was removed even more gradually. A second set of experiments involved studies of the orientation of the receptors on the reconstituted liposomes. Twothirds of the receptor-bound IgE dissociated at acid pH in the experiment illustrated; other experiments yielded even higher values. These should be taken as lower limits since some of the dissociated IgE may have interacted with the acid-modified liposomes (9). These results are consistent with our other findings on the orientation of the reincorporated receptors ( 1). Thus, 70-80% of such liposomes bound to beads containing antigen to which the IgE was directed. Likewise, tryptic digestion showed that 90% of the IgE was susceptible to cleavage. All three approaches suggest that the majority of the reincorporated IgE-receptor complexes were properly oriented. REFERENCES
5. 6. 7.
4 In instances where the proteinlipid ratio can be markedly increased and steeper sucrose gradients are employed, centrifugations of four to six hours may be adequate. Nevertheless, the time is markedly longer than that required using the Airfuge.
8. 9.
Rivnay, B., and Metzger, H. (1982) J. Eiol. Chem. 257, 12,800-12,808. Isersky, C., Kulczycki, Jr., A., and Metzger, H. ( 1974) J. Immunol. 112, 1909-1919. Kulczycki, Jr., A., and Metzger, H. (1974) J. Exp. Med. 140, 1676-1695. Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1965) Biochem. J. 89, 114-123. McConahey, P. J., and Dixon, F. J. (1966) Int. Arch. Allergy Appl. Immunol. 29, 185- 189. Eccleston, E., Leonard, B. J., Lowe, J. S., and Welford, H. J. (1973) Nature New Biol. 244, 73-76. Huganir, R. L., Schell, M. A., and Racker, E. (1979) FEBS Lert. 108, 155-160. Kagawa, Y., and Racker, E. (197 1) .r. Biol. Chem. 246, 5477-5487. Vandenbranden, M., DeCoen, J. L., Jeener, R., Kanarek, L., and Ruyschaert, J. M. (1981) Mol. Immunol. l&621-631.
BIOCHEMISTRY
130,
514-520
(1983)
Use of the Airfuge for Analysis and Preparation of Receptors Incorporated into Liposomes: Studies with the Receptor for lmmunoglobulin E BENJAMIN RIVNAY’ AND HENRY METZGER~ Section on Chemical Immunology, Arthritis and Rheumatism Branch, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205 Received September 23, 1982 A Beckman Airfuge has been employed for studying the interaction between lipids and the receptor for immunoglobulin E (IgE). For analytic experiments, samples were applied underneath a discontinuous sucrose gradient. After a 30-min centrifugation in a fixed-angle rotor, liposomes floated toward the top of the gradient whereas unincorporated receptor-IgE complexes remained at the bottom of the tube. Liposomes with incorporated receptors were also efficiently separated in the ACR-90 preparative rotor. These methods of “Airfuge flotation” can provide useful adjuncts to more traditional methods for density-gradient centrifugation especially when rapid analysis of small samples is desired.
In most work on the reconstitution of membrane proteins, a mixture of lipids, protein, and detergent is dialyzed to remove the detergent. Whereas the protein is often efficiently incorporated into the liposomes formed in this way, this is not always so. It therefore becomes necessary to analyze the dialyzed mixture or to separate the unincorporated material for preparative purposes. This proved to be the case in our investigations on the receptor for immunoglobulin E (IgE)3 (1). In those experiments we analyzed the mixtures by the traditional procedure of sucrose-density-gradient centrifugation in swinging-bucket rotors utilizing an ultracentrifuge. During the course of those studies we explored the possibility of using much shorter centrifugations in the Beckman Airfuge as an adjunct or alternative to such analyses. In this paper we describe the procedures which we found to be useful. ’ Present address: Weizmann Institute of Science, Rehovot, Israel. 2 To whom correspondence should be addressed at: Building 10, Room 9N-2 18, National Institutes of Health, Bethesda, Md. 20205. r Abbreviation used: IgE, immunoglobuhn E. 0003-2697183
$3.00
Copyright Q 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
514
MATERIALS
AND METHODS
Chemicals. Rat immunoglobulin E was isolated from as&tic fluid of tumor IR 162 as described by Isersky et al. (2) and by Kulczycki and Metzger (3), and iodinated by the chloramine-T method (4, 5) using Na1251 (Amersham, Arlington Heights, Ill.). Cytochrome c from horse heart, Type III (No. C-2506), 1-cu-phosphatidylcholine from soybean (No. P-3644), 1-O-n-octyl-P-D©ranoside (octylglucoside, No. O-800 l), iodoacetamide (No. I-6 125), phenylmethylsulfonyl fluoride (No. P-7626), aprotinin from bovine lung (No. A-60 12) and pepstatin (No. P-4265) were purchased from Sigma (St. Louis, MO.). Leupeptin was brought from Boehringer-Mannheim Biochemicals (Indianapolis, Ind.) and sucrose (density gradient grade) from Schwartz/Mann (Orangeburg, N. Y.). The buffer used throughout this study was Tris-saline (15 mM Tris-HCl, pH 7.6, 5 mM KCl, and 125 mM NaCl). Liposomes. Sonicated liposomes were prepared by thoroughly drying a chloroform solution of bis( [ 1-14C]palmitoyl)phosphati-
AIRFUGE
FLOTATION
dylcholine and soybean lecithin, swelling in Tris buffer at a concentration of 50 mg/ml, and sonicating with the probe of a sonicator (Model W225R, Heat Systems, Plainview, N. Y.) at 70% output on ice. The supernatant was used after centrifugation at 3 1,OOOg,, at 4°C for 30 min. Liposomes prepared by the dialysis method were prepared in a similar way to that described in the section on Reconstitution except that the mixture did not contain cell extract or cytochrome c. Receptor solubilization. Rat basophilic leukemia cells (2H3 subline (6)), to which 1251IgE was bound, were solubilized with octylglucoside at final concentrations of 5 X 10’ cells/ml (25 nM in receptor-bound IgE) and 40 mM octylglucoside, respectively. Six inhibitors of proteolysis were added: 20 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 25 pg/ml aprotinin, 3 pg/ml pepstatin, and 1 pg/ml leupeptin. The extract was incubated 10 min on ice, centrifuged for 1 h at 3 1,OOOg,, at 4°C and the supernatant used immediately or stored at -80°C. The concentration of endogenous phospholipids was -2
rnM.
Reconstitution. Eighty microliters of detergent extract containing receptor-IgE complexes (cf. Receptor Solubilization) was mixed with exogenous lipids (soybean lecithin liposomes) and additional detergent to final concentrations of 5 nM bound IgE, 16 mM soybean lecithin, 200 mM octylglucoside, and 2 mg/ml cytochrome c (as carrier), and incubated on ice for 2 h. The detergent was removed by dialysis against 500 vol of Tris buffer with 3-4 daily changes for 3-4 days. Acid dissociation of IgE. IgE was dissociated from the reconstituted liposomes by acid treatment. Sixty-four microliters of the reconstituted liposomes in 15 mM Tris-HCl, pH 7.5, was incubated with 20 ~1 of 100 mM glycine . HCl, pH 2.35, (final pH 3) for 1 min, and then neutralized with 73 ~1 of a 150 mM Tris buffer (pH 8.1) containing 60% sucrose and 10 pg/ml unlabeled IgE (final pH 7.5). All incubations were done on ice. Gradient centrifugation. Stock solutions of 5, 10, 20,25,27, and 60% sucrose (w/w) were
OF
515
LIPOSOMES
prepared and checked by refractive index measurements on an Abbe-type refractometer (Bausch & Lomb, Rochester, N. Y .). Stepwise gradients of 4-5 layers were sequentially applied in Beckman Airfiuge test tubes (cellulose nitrate No. 339647 (4.8 X 19.9 mm) or Ultraclear No. 344718 (5 X 20 mm), Beckman, Palo Alto, Calif.) for a total gradient volume of 145- 150 ~1 (excluding the samplecontaining layer of 30-40 ~1). The compositions of gradients used in individual experiments are detailed in the legends to the figures. Since differences of 50-60 mg in tare weight were often found between individual test tubes, pairs of test tubes were weighed and matched within l-2 mg as a precaution to minimize rotor wobbling during acceleration and deceleration. In order to avoid turbulence and intermixing of the sucrose layers during construction of the gradient, the test tubes were frozen on dry ice for 1 min after each addition of a new layer. The work was facilitated by the use of a tube holder consisting of an aluminum block which was placed in granular dry ice. Twelve gradients of five steps were readily prepared in less than 15 min. The gradients were thawed and the specimen applied with a Hamilton syringe prior to the run. Centrifugation was carried out in a cold room (4°C) at top speed (30 psi) and operated from a wall outlet of pressurized air. Fractions of 25 ~1 were collected from the top with a pipettor held parallel to the tube, and with minimal insertion of the tip into the gradient. Counting. ‘251 counting was performed in a Beckman Gamma 8000 (Palo Alto, Calif.). 14Cradioactivity was counted in Aquasol (New England Nuclear, Boston, Mass.) in a Mark III scintillation counter (Tracer, Elk Grove Village, Ill.) with the 14C channel set to 65400 keV. RESULTS
Analytic separations. Density-gradient centrifugations are commonly performed in rotors with swinging buckets thereby permitting the gradient vector to remain parallel to the sides of the tube throughout the run. In a fixed angle rotor this relationship is not maintained
RIVNAY
516
AND METZGER
thereby potentially causing destruction of the gradient. We tested to see if this would vitiate the possibility of using the fixed angle (0 = 18 “) rotor of the Airfuge. Figure 1 shows the results of two experiments employing duplicate discontinuous sucrose gradients which were centrifuged for 30 min (Fig. IA) and 40 min (Fig. lB), respectively. The patterns of the recovered gradients are superimposed over the applied gradients. As can be seen, the average slope of the recovered gradients was only about one-half of that of the gradient prior to centrifugation. However, it is apparent that there FRACTION
12345676 I
r
xl-
I
FRACTION
I
A
20-
2oLP-
i
FIG. 2. Liposome migration in Airfuge gradients. Four gradients containing 35 ~1 30%, 45 ~1 25%, 45 ~1 208, 30 ~1 IO%, and 20 pl 5% sucrose were prepared. In (A) the liposomes were contained in the top fraction. In (B) 20 ~1 of W-labeled liposomes in 30% sucrose were inserted as the lower part of the bottom fraction. One gradient in each set contained sonicated liposomes (0 - - - 0), the other one contained liposomes formed by detergent dialysis (0 0). Gradients were run and collected as detailed under Materials and Methods and counted in the scintillation counter. Vertical bars represent the radioactivity which remained in the empty test tubes: open bars, ?-lipids; filled bars, izsI-IgE.
was a monotonic increase in the density of the specimens recovered by taking successive L-J samples from the top of the tube. Moreover, the results from the duplicate tubes showed excellent reproducibility. No change in the VOLUME (pL1 slope of the recovered gradient was observed FIG. 1. Stability of stepwise gradients centrifuged in the when the tubes were left in a vertical position A- 100 (fixed angle) rotor in the Airfuge. (A) Two gradients containing 50 ~1 of 296, IO fll 25%, 30 ~1 20%, 45 ~1 for 1 h after centrifugation. The last fraction IO%, and 40 ~1 5% sucrose were. run for 30 min. 25-~1 usually had a slightly lower density than the fractions were collected from the top, and the refractive penultimate one (Fig. 1), probably due to slidindices measured and translated to sucrose concentrations. One gradient (-) was analyzed immediately, and ing of some of the top fraction down the sides the other (- - -) one hour later. (B) Two gradients (solid of the tube during removal of successive fracand dashed lines) with 35 ~1 of 30%, 40 pl 26%, 35 pi tions. We assessed whether it would be better 25%, 30 pl20%, 25 ~1 5% sucrose, and 20 ~1 Tris buffer to apply the samples on top of, or beneath, were run for 40 min. Fractions were immediately col- the gradient. Figure 2 shows the distribution lected from both gradients. (O 0) pattern of the original gradient. Arrows indicate the lower density of the of two types of liposomes when applied to the volume remaining for the partial 8th fraction. See also top or underneath identical gradients. Neither Fig. 4 for the choice of centrifugation period. the liposomes prepared by sonication (small, 10
AIRFUGE
FLOTATION
unilamellar) nor those prepared by detergent dialysis (large uni- and multilamellar) sedimented when applied to the top of the gradient (Fig. 2A). However, when the sample was applied under the gradient a differential flotation was observed (Fig. 2B). The liposomes formed by dialysis which have a lower density because they contain a large internal volume of sucrose-free solvent, floated to the top of the gradient whereas the sonicated vesicles floated only halfway up the gradient. A comparable experiment employing unbound IgE showed the expected result. When applied under the gradient, the IgE remained in the bottom fractions; when applied to the top of the gradient, it failed to sediment significantly during the 30-min centrifugation. The combined results with the liposomes and the IgE indicated that in short centrifugations the specimens had to be applied underneath the gradient in order to separate liposome-bound from unincorporated IgE-receptor complexes. We refer to this procedure as “Airfuge flotation.” After numerous experiments with patterns similar to that shown in Fig. 2B, we chose to consider the material in the top four fractions as representing liposome-associated components. Recoveries of 14C-lipids in these fractions routinely ranged between 65 and 85% of the applied amount; the remainder was found in the bottom fractions and in the empty test tube (~9%). Several examples of Airfuge flotation will now be given. In one experiment we wished to determine if the speed of removal of the detergent would affect the incorporation. We prepared a detergent extract of cells to which ‘251-IgE had been bound. The extract was supplemented with asolectin, octylglucoside, and cytochrome c (as carrier). One aliquot was thoroughly dialyzed (see Materials and Methods): the other was passed over a column of Sephadex G-25 (medium) and the fractions from the void volume were collected. Figure 3 depicts the analysis of the two separations. With the dialyzed sample -35% of the ‘251labeled IgE and -83% of the i4C-lipids were
517
OF LIPOSOMES
1
3
5
7
FRACTION
FIG. 3. Airfuge flotation of vesicles reconstituted with crude extracts of cells containing &-receptor complexes and soybean lecithin. The 1251-IgE-receptors were solubilked as detailed under Materials and Methods mixed with additional octylghtcoside (200 mM), soybean lecithin (16 mM), and with cytochrome c (2 mg/ml) as carrier, and incubated 2 h on ice. Two hundred microliters of the mixture was dialyzed thoroughly (A), and 90 ~1 was passed through a 1.5 X 13-cm column of Sephadex G-25 medium (B). The reconstituted material (from (A) and (B)) was made 30% in sucrose, and 35 ~1 floated in duplicate gradients composed of 45 ~125%, 40 ~120%, 25 ~1 IO%, and 30 ~1 5% sucrose. After 30 min centrifugation, the gradients were analyzed as described. Bars represent Wlipids (0) or ‘Z51-IgE (m) counts remaining in the empty test tubes. The combined gradients in panel (A) contained 11,700 cpm of 14Cand 8860 cpm of ‘251;and in (B), 7050 and 5 100, respectively.
recovered in the top four fractions of the gradient (Fig. 3A). With the sample filtered over Sephadex the comparable fractions contained -76% of the lipids but only - 19% of the IgE. If we assume that those lipids which were not recovered in the top four fractions nevertheless represented liposomal material, then the “corrected” values for the liposome-associated h&receptor complexes are 42 and 25%, respectively. On the basis of these results we adopted dialysis as the method of choice. In the initial phase of this study, a 30-min centrifugation was chosen arbitrarily. In order to check whether this time was appropriate, a preparation which contained approximately
RIVNAY
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AND METZGER
equal amounts of free and liposome-associated receptor-IgE complexes was centrifuged during 10, 20, or 40 min at 30 psi. Though flotation was somewhat better after 40 min than after 20 min (Fig. 4), the relative recoveries of the i4C-lipids and of the ‘251-IgE counts (not shown for sake of clarity) in the top four fractions were very similar. After 10 min of centrifugation the liposomes migrated only half way and the separation from the unincorporated complexes was poor. When two Airfuges are connected in tandem, thereby permitting 12 samples to be centrifuged simultaneously, the air pressure (obtained from a wall outlet) may be somewhat lower (e.g., 20-25 psi). This in turn reduces the speed of centrifugation - 10 percent. We checked the effect of this variable. Identical gradients were centrifuged for 40 min with an air pressure of 20 psi (instead of 30 psi). Under these conditions the peak of the liposomes was at fractions 2 and 3, still well separated from the unincorporated receptors. Preparative separations. We tested to see if preparative separations could be performed by using the ACR-90 rotor (Fig. 5A). This
G $40
U-JO 03 his ug :! 5
E
40 min. -20 min.
10 min.
20
r!iiiil 1
3
5
7
FRACTION
FIG. 4. Length ofcentrifugation and liposome flotation. IgE-receptor complexes were reconstituted by dialysis as described under Materials and Methods and analyzed on gradients containing 40 ~1 27%, 35 ~1 25%, 30 ~1 20%, 25 ~1 5% and 20 ~1 0% sucrose. Identical samples were made 30% in sucrose, immediately centrifuged for the indicated periods of time, and analyzed as described under Materials and Methods. ‘*‘I counts recovered in the top 4 fractions were 35, 36, and 21% at 40, 20, and 10 min, respectively. The corrected values were calculated to be 41, 42, and 35% in the respective gradients. Less than 0.5% of the i4C cpm were associated with the empty test tubes.
A
liner adapter
outer inner
chamber rotor
outer
1
ring
chamber
base
chamber
3
5 FRACTION
7
FIG. 5. Isolation of liposomes with the ACR-90 rotor. A specimen that has been reconstituted from an octylglucoside-solubilized extract of cells with soybean lecithin (see Materials and Methods) was made 30% in sucrose and floated in the small liner (2.4 ml) of the ACR-90 rotor. (A) Schematic cross section of the ACR-90 rotor. 1 ml of 16% sucrose was injected into the outer chamber (1.4 ml) of the liner, and 0.7 ml of the specimen was injected underneath, causing an overtlow of the lighter cushion into the inner chamber (- 1 ml). The volume occupied by the specimen is marked by the horizontal dotted line. The inner chamber was then filled with 16% sucrose, the lid closed, and the rotor run for 75 min. The contents of the inner chamber were removed and fractions of 300 ~1 were collected from the bottom of the outer chamber. All additions and withdrawals were performed with a I-ml syringe attached to a blunt 23G needle. Horizontal bar in top right comer = 1 cm. This Figure is modified from Beckman Manual No. AF-TBOOZD, Feb. 1979. (B) Distribution of radioactivity in the inner chamber and the four fractions of the outer chamber. (C) Airfuge flotation analysis of the unfractionated material performed with the A-100 rotor. (D) Analysis of a sample from the inner chamber performed as in (C). Open symbols or open bars, 14C-lipids; filled circles and bars, ‘*‘IIgE. Bars in panels (C) and (D) represent material remaining in the empty test tubes.
AIRFUGE
FLOTATION
rotor was designed for clarification of lipemic sera and separation of chylomicrons. A reconstitution mixture was dialyzed and made 30% in sucrose. It was applied in the bottom of the outer chamber of the rotor liner under a layer of 16% sucrose as detailed in the legend to Fig. 5. The rotor was accelerated slowly (46 min) in order to avoid intermixing of the two sucrose layers and was then spun for 75 min. The entire contents (- 800 ~1) from the inner chamber were withdrawn without removing the rotor from the Airfuge, and then four fractions were collected from the bottom of the outer chamber. Analysis of the specimens (Fig. 5B) showed that more than 90% of the 14C-lipids were recovered in the inner chamber and about 50% of the 1251-IgE was found in this pool. Airfuge flotation in the A100 analytic rotor was performed on the unseparated mixture (Fig. 5C) as well as on the material recovered from the inner chamber (Fig. 5D). It can be seen that the unfractionated material (Fig. 5C) showed a distribution of 1251-IgE-receptor complexes consistent with the results obtained in the preparative run; the corrected value for incorporation was 46%. On the other hand, the sample from which the unincorporated receptors had been removed in the preparative centrifugation (Fig. 5D) now shows a virtually perfect concordance between the distribution of radioactivity associated with the lipids and that associated with the IgE-receptor complexes. The percentage of receptor complexes associated with the liposomes was calculated to be 98%. Airfuge flotation was also used to characterize the incorporated IgE-receptor complexes in terms of exposure to the external medium. Exposure of cells to acid (pH 3) rapidly dissociates the bound IgE (3). We have measured the dissociable fraction of IgE on the reconstituted liposomes in order to assess the amount putatively exposed on the outer surface of the liposomes. Vesicles containing IgE-receptor complexes were preparatively separated from the unincorporated material as described for Fig. 5 and the liposomes from
519
OF LIPOSOMES
the inner chamber were recovered. One aliquot was treated with acid for 1 min, and then was neutralized with Tris buffer containing unlabeled IgE (Fig. 6B). A control aliquot was treated with the same solutions as above, but these had been premixed (Fig. 6A). Only 37% of the labeled IgE remained associated with the liposomes after the acid treatment whereas the control sample showed 9 1% association. This suggested that approximately two-thirds of the complexes were properly oriented. We noted that the liposomes became more turbid upon acidifying and remained so after neutralization. It is also apparent that the treated liposomes migrated differently than the shamtreated material in the gradients (of Figs. 6A and B). It is possible that the acid pH’s caused the liposomes to fuse with each other but we have not checked this. Parallel experiments, in which rebinding of IgE to acid-dissociated complexes was studied (data not shown), showed quantitative rebinding of new IgE to the vacant receptors. This excluded the possibility that the receptors themselves had been detached from the liposomes at the acid pH.
1
3
5
7
FRACTION
FIG. 6. Acid-induced dissociation of IgE from reconstituted liposomes. (A) Control: separated liposomes were incubated with premixed acid and neutralizing buffers (Materials and Methods), and then analyzed by Airfuge flotation. (B) Acid treatment: hposomes and buffers like in (A) were mixed sequentially, the neutralizing buffer was added 1 min after the acid buffer, and the mixture was then analyzed as in (A). 0 - - - 0, “C-lipids; 00 ‘251-IgE. Filled and empty bars represent the “‘1 and I& counts in the empty test tube, respectively.
RIVNAY
520
AND METZGER
DISCUSSION
We have presented a rapid semiquantitative method for analyzing protein-liposome interactions. The method is based on the differential flotation of the constituents in the mixture on sucrose gradients, utilizing a commercially available small air-driven centrifuge (Airfuge). Both analytical runs of six specimens of ~50 ~1 and preparative separations of ~750 ~1 can be performed, utilizing the A100 and ACR-90 rotors, respectively. The procedure yields reproducible separations. Duplicate samples generally give distributions of the components within 5% of each other, when run on the same gradients in different centrifugations. The method is economic in both price and space, by the nature of the equipment employed. It also uses little material and is rapid. Many steps in the procedure take less time than the parallel ones in the full-size ultracentrifuge. The most important difference is the time of centrifugation. We routinely used 30 min per run, which can be compared to the 18- to 36-h runs required in our earlier work using standard methods. Thirty centrifugations (180 tubes) can, in theory, be analyzed during the period of one overnight run in the ultracentrifuge; in practice, 4-5 centrifugations (24-30 tubes) were analyzed in one day by one person.4 We have illustrated the usefulness of the method with two sets of experiments involving the reconstitution of the @-receptor complexes. In the first, we demonstrated that dialysis was preferable to gel filtration for removal of the detergent. Earlier reconstitution studies with other systems (7,8) indicated that,
with Sephadex filtration or rapid dilution, less or improper reconstitution occurred. Failure to achieve efficiencies higher than 40-50% (Figs. 3 and 5) is related, in the case of the IgE-receptor system, to the nature of the detergent (octylglucoside) and the lipid (asolectin) used (l), and no improvement was observed when the detergent was removed even more gradually. A second set of experiments involved studies of the orientation of the receptors on the reconstituted liposomes. Twothirds of the receptor-bound IgE dissociated at acid pH in the experiment illustrated; other experiments yielded even higher values. These should be taken as lower limits since some of the dissociated IgE may have interacted with the acid-modified liposomes (9). These results are consistent with our other findings on the orientation of the reincorporated receptors ( 1). Thus, 70-80% of such liposomes bound to beads containing antigen to which the IgE was directed. Likewise, tryptic digestion showed that 90% of the IgE was susceptible to cleavage. All three approaches suggest that the majority of the reincorporated IgE-receptor complexes were properly oriented. REFERENCES
5. 6. 7.
4 In instances where the proteinlipid ratio can be markedly increased and steeper sucrose gradients are employed, centrifugations of four to six hours may be adequate. Nevertheless, the time is markedly longer than that required using the Airfuge.
8. 9.
Rivnay, B., and Metzger, H. (1982) J. Eiol. Chem. 257, 12,800-12,808. Isersky, C., Kulczycki, Jr., A., and Metzger, H. ( 1974) J. Immunol. 112, 1909-1919. Kulczycki, Jr., A., and Metzger, H. (1974) J. Exp. Med. 140, 1676-1695. Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1965) Biochem. J. 89, 114-123. McConahey, P. J., and Dixon, F. J. (1966) Int. Arch. Allergy Appl. Immunol. 29, 185- 189. Eccleston, E., Leonard, B. J., Lowe, J. S., and Welford, H. J. (1973) Nature New Biol. 244, 73-76. Huganir, R. L., Schell, M. A., and Racker, E. (1979) FEBS Lert. 108, 155-160. Kagawa, Y., and Racker, E. (197 1) .r. Biol. Chem. 246, 5477-5487. Vandenbranden, M., DeCoen, J. L., Jeener, R., Kanarek, L., and Ruyschaert, J. M. (1981) Mol. Immunol. l&621-631.