Biochem. J. (1975) 152, 267-270

267

Printed in Great Britain

Reconstitution of Pig Lymphocyte Plasma Membranes from Solubilized Components, with Particular Reference to Membrane-Associated Tinmunoglobulins By STEPHEN I. CHAVIN* and ANDREW HOLLIMAN A.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, U.K. (Received 27 May 1975)

Plasma membranes of lymphocytes obtained from pig mesenteric lymph nodes were reconstituted after solubilization with bile salts. The proportion by weight of immunoglobulin in the reconstituted membrane was no greater than about 5-10% of that in the original membrane. Possible reasons for the low reincorporation of immunoglobulin are discussed.

Although serum immunoglobulins have been incorporated into model membrane systems (Marinetti & Pettit, 1968; Weissman et al., 1974), the nature of these interactions and their relevance to the function of the naturally occurring membraneassociated immunoglobulin is unknown. Membranous structures can re-form readily from dispersions of their components on removal of the solubilizing agent (Razin, 1972). Although in some instances there was a marked loss of biological activity (Slack et al., 1972), or interactions occurred between the reconstituted protein and phospholipid which appeared to be quite different from those in the original membranes (Metcalfe et al., 1971; Tillack et al., 1970), progress has been made in the reconstitution of membranes with functional activity (Racker & Stoeckenius, 1974; Warren et al., 1974). We report here studies on attempts to reincorporate membraneassociated immunoglobulins into reconstituted lymphocyte plasma membranes. Materials and Methods Preparation of membranes Membranes were prepared from intact mesenteric lymph nodes of young pigs by the method of Allan & Crumpton (1970) as modified in our laboratory (Chavin et al., 1975). Reconstitutions

Reconstitutions were carried out on membranes solubilized with deoxycholate by the method of Changeux et al. (1972), except for the isolation of the reconstituted membrane. Reconstitutions were also carried out by using sodium cholate [purchased as cholic acid; Sigma (London) Chemical Co., Kingston-upon-Thames, Surrey KT2 7BH, U.K.] to * To whom reprint requests should be addressed. Present address: Medical Research Council Laboratory, Oxford Haemophilia Centre, Churchill Hospital, Headington, Oxford OX3 7LJ, U.K.

Vol. 152

dissolve the membranes. A stock solution of 21 % (w/v) was prepared by carefully dissolving a suspension of the cholic acid and adjusting the pH to 7.4 (at 21 C), with 6 M-NaOH. The membrane was pelleted at lOOOOOgav for 30min, the pellet suspended in 0.8ml of 1.5M-Tris-HCl (1.SM-Tris adjusted to pH 7.4 with 6M-HCI), and dissolved by adding 0.8ml of 21 % (w/v) cholate solution. The ratio cholate/protein was usually about 35:1 (w/w). The clear solution was stirred at 21°C for 30min, diluted with 7.2ml of water, and stirred for an additional 90min. The solution was centrifuged at 100000 ga,. for 30min and the supernatant placed in 0.63cm (kin) Visking dialysis tubing and dialysed against 2 litres of solution [200mM-sucrose-50mM-MgCl2SOmM-Tris-HCl (pH 7.4, 50mM-Tris adjusted to pH 7.4 with 6M-HCI)] for 17h at 4°C, and a further 2 litres for an additional 4h. At the end of the dialysis the cloudy suspension was centrifuged in a Spinco L2 65B centrifuge, in an SW 50L rotor at 105000g,v. for 60min at 4°C. The opalescent supernatant (unreconstituted protein) was decanted and the pellet dispersed and mixed first with one drop of 80% (w/v) sucrose and then with approx. 0.5ml of 50 % (w/w) sucrose. A linear sucrose gradient, 50-20 % (w/w), was formed over the membrane sample at 4°C. All sucrose solutions were in 50mMTris-HCI buffer, pH 7.4. The gradients were centrifuged in a Spinco SW 50L rotor at 105000g,V for 18 h at 4°C. The membranes were recovered as a single band of white particulate material of varying width, and there was usually a small amount of low-density material at the top of the gradient. Small portions of sucrose were carefully removed with a Pasteur pipette from immediately above and immediately below the visible membrane band and the refractive index was measured at room temperature with a refractometer. The sucrose concentrations and densities were calculated from data in the International Critical Tables and the Handbook of Chemistry andPhysics.

268

Other methods Polyacrylamide-gel electrophoresis (Neville & Glossmann, 1971) and immunological procedures (Heremans, 1971) were performed as reported (Chavin et al., 1975). Protein was determined by the method of Lowry et al. (1951), except that Mg2+ was included in blanks and standard bovine serum albumin solutions when the unknowns contained this cation. Material for electron microscopy was fixed as a suspension in 4 % (v/v) glutaraldehyde (reagent grade; Koch-Light Laboratories, Colnbrook, Bucks, U.K.) in 0.1 M-Na2HPO4 buffer (adjusted to pH 7.2 with concentrated H3PO4) for 30-60min at room temperature, and then pelleted at lOOOOOgav, for 30min. The pellet was resuspended in the phosphate buffer without glutaraldehyde and re-centrifuged as above. Post fixation was with 1 % OS04 in 0.1 M-veronalacetate buffer, pH 7.2, for 60min, followed by a water rinse. The pellets were stained in aq. 2 % (w/v) uranyl acetate for 60min, dehydrated in an alcohol series followed by a propylene oxide soak, and finally embedded in Araldite. Sections were stained as necessary with uranyl acetate [saturated in 50% (v/v) alcohol] and lead citrate. Results and Discussion Choice ofdetergent We have used the sodium salts of deoxycholate or cholate to dissolve the membranes. Although both bile salts are effective for solubilizing the membranes, neither deoxycholate (Crumpton & Parkhouse, 1972) nor cholate (S. I. Chavin, unpublished work) significantly affects the antigenicity of immunoglobulin or interferes with antigen-antibody interactions at the concentrations used in the present experiments. The critical micellar concentration of deoxycholate is about 10mM (0.39%, w/v), whereas that of cholate is about 45mm (1.89 %, w/v) (Kagawa, 1972). Thus although cholate must usually be used at a higher concentration than deoxycholate for comparable effects, the former has two major advantages: (1) the micellar molecular weight of cholate (819) is lower than that of deoxycholate (1963) and therefore the cholate can be more rapidly removed by dialysis; (2) solutions of cholate do not gel and precipitate as do those of deoxycholate.

Reconstitution The following results are from experiments using cholate unless otherwise stated. About 90-95% of the membrane protein was soluble, and the cholate solution contained the same relative amounts of all the major polypeptide chains as the original membrane dissolved in sodium dodecyl sulphate [Plate 1(A), samples e and a respectively]. The pellet of

S. I. CHAVIN AND A. HOLLIMAN

cholate-insoluble material was extremely difficult to dissolve in sodium dodecyl sulphate-Na2CO3 solution and no further work was done with this fraction. The yields of reconstituted membranes (measured as protein) varied between 20 and 40 % of the protein in the cholate solution. The reason for the variable yields is not known. The reconstituted membranes were usually purified on continuous sucrose gradients because we found that the separated proteins and phospholipids could aggregate on removal of detergent, and we did not want the membranous material to be contaminated with aggregates of protein or phospholipid alone. The reconstituted membranes were recovered as a single broad band, which often was skewed towards one or the other extreme. The original membrane was recovered as a narrower band with only minimum trailing on either side of the main band. The relative densities and refractive indices from a number of different experiments are listed in Table 1. It is clear that the reconstituted material was more heterogeneous in density than was the original membrane, and that the re-formed membranes could be either more or less dense than the original ones. The most probable explanation for this heterogeneity is variation in protein/phospholipid ratio among the vesicles, but an additional explanation might be the trapping of soluble and/or particulate material within the vesicles.

Electron microscopy Gradient-isolated reconstituted membranes and original membranes were indistinguishable from one another by electron microscopy (Plates 2a and 2b). Both consisted of closed vesicles variable in size, but with an average diameter of approx. 0.050.1,um (500-1000A). Occasional vesicles contained amorphous inclusions. Material reconstituted from membrane protein alone formed amorphous aggregates in which no definite structure could be discerned, whereas that from the separated membrane phospholipids gave multilayered 'onion-skin' structures typical of phospholipid bilayers. Membrane fragments could not be identified morphologically in the unreconstituted protein fraction by electron-microscopic examination of negatively stained preparations. We have no further information on the molecular form of this unreconstituted material, e.g. whether the proteins are in true solution or whether they are associated with phospholipids in micelles alone or with bound detergent.

Polyacrylamide-gel electrophoresis In sodium dodecyl sulphate-polyacrylamide gels, the reconstituted membranes showed marked aggregation and streaking, especially among the 1975

The Biochemical Journal, Vol. 152, No. 2

Plate 1

(A)

Mol. wt. 1.

470 000

2. 3.

420 000 3 00 0 0 0 140 000 98 000

4. 5.

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EXPLANATION OF PLATE I

Polyacrylamide-gel electrophoresis oforiginal and reconstitutedpig mesenteric-node lymphocyte plasma membranes (A) and the presence of membrane-associated immunoglobulin in reconstituted pig mesenteric-node lymphocyte plasma membrane demonstrated by radial immunodiffusion (B) I(A). (a) Original plasma membrane, prepared as described in text; about 66pg of protein. (b) Reconstituted membrane, pelleted but not isolated on a gradient; protein not measured. (c) Reconstituted membrane, gradient isolated from a portion of the pellet in sample (b); protein not measured. (d) Unreconstituted protein, remaining in supernatant after reconstitution mixture was centrifuged at I00000gav. for 60min; about 80ug of protein. (e) Cholate solution of original plasma membranes. All of the samples were delipidated with chloroform-methanol (2: 1, v/v) to separate the protein from phospholipid and to remove detergent when necessary, and were then dissolved in sodium dodecyl sulphate-Na2CO3. 1(B). The membrane was reconstituted from cholate solution, but was not gradient isolated. The agar contains 0. 14/ (v/v) of sheep anti-(pig IgG), reacting with light and y chains. Row 1: pig IgG, (a) 50,ug/ml, (b) 25,g/ml, (c) 50,pg/ml, (d) 25,pg/ml. Row 2: (a) reconstituted membrane, dissolved 14%. (w/v) sodium cholate, about 35 mg of protein/ml; (b) the left well contains same membrane as in row 2 (a), and the right well contains pig IgG (100,g/ml); (c) and (d), original membrane reisolated on sucrose density gradient (24mg of protein/ml). The plate was incubated for 11 days and photographed after washing, drying and staining. The reconstituted samples show a faint and diffuse precipitin ring, which interrupts and distorts the ring due to the IgG.

S. I. CHAVIN AND A. HOLLIMAN

(Facing p. 268)

Plate 2

The Biochemical Journal, Vol. 152, No. 2

(b

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EXPLANATION OF PLATE 2

Electron micrographs oforiginal (a) and reconstituted (b) pig mesenteric-node lymphocyte plasma membrane

2(a) Magnification of original electron microscope plate x 30000; total magnification of this plate is x51 000. 2(b) Magnification of the original electron microscope plate, x40000; total magnification of this plate is x 68 000.

S. I. CHAVIN AND A. HOLLIMAN

RECONSTITUTION OF SOLUBILIZED LYMPHOCYTE PLASMA MEMBRANES

269

Table 1. Relative densities oforiginal and reconstitutedplasma-membrane preparations The gradients were sampled with Pasteur pipettes, at the upper and lower extremes of the visible membrane band before the membrane material was removed. The refractive indices were determined at room temperature with a refractometer; the headings 'upper' and 'lower' refer to the respective limits of the membrane band. The refractive indices are given as the mean + S.D for each type ofmembrane. The range of densities was calculated from the means of the refractive indices, by using data in the Handbook of Chemistry and Physics, 52nd edn., p. D 221. No. Refractive index of Density Lower limit Upper limit Membrane samples (g/il) 3 1.3816+0.0042 1.3926+0.0036 1.128-1.157 Original Reconstituted 1.3689+0.0022 1.3933±0.0089 9 1.094-1.159 Deoxycholate 15 1.127-1.185 Cholate 1.3812±0.0085 1.4032±0.0126

polypeptides of higher molecular weight, compared with the original membranes and with the cholate and deoxycholate solutions of these membranes. Certain reconstituted preparations were less aggregated than others and appeared to contain the major polypeptide chains in approximately the same relative proportions as in the original membranes [Plate 1(A), samples c and a respectively]. The tendency for the reconstituted membranes to aggregate could be diminished by dialysis against EDTA, suggesting that the Mg2+ in the reconstitution buffer might be partially involved. We do not think that proteolytic or other types of degradation are responsible for the poor electrophoretic resolution because: (1) storage of the membrane in cholate solution for as long as 1 week did not affect the polypeptide-chain pattern; (2) the addition of proteolytic enzymes to a sodium dodecyl sulphateNa2CO3 solution of the membranes and incubation before electrophoresis caused a marked loss of polypeptide chains larger than around 46000 daltons, but did not result in aggregation or streaking; (3) the addition of phenylmethanesulphonyl fluoride immediately after disruption of the cells, to inhibit endogenous proteolytic activity, did not affect the amount of aggregation and streaking. Reincorporation of membrane-associated immunoglobulin Several samples of reconstituted membranes were examined for their immunoglobulin content. Two of these (cholate procedure, not gradient isolated) gave a faint, diffuse precipitin ring which could not be accurately measured [approx. 0.1 % (w/w) immunoglobulin for the sample in Plate 1(B)]. A third sample (deoxycholate procedure, gradient isolated) gave a sharp precipitin ring, with an estimated immunoglobulin concentration of about 0.06% (w/w). Since the original membranes contain 1% (w/w) immunoglobulin (both IgG* and IgM) * Abbreviations: IgG, immunoglobulin G; IgM, immunoglobulin M (macroglobulin). Vol. '152

(Chavin et al., 1975), the reconstituted membranes contain no more than 5-10% of the original amount. The small amounts of immunoglobulin in the reconstituted membrane are unlikely to be due to passive trapping of soluble immunoglobulin within the re-formed vesicles, as shown by the following calculations. Before reconstitution a cholate solution with a protein concentration of 400pug/ml will contain about 40ng of immunoglobulin in 10pl; 1000l g of reconstituted membrane protein with 0.1 % (w/w) immunoglobulin will have 10OOng of immunoglobulin in a volume of 10-20,u1 (after centrifugation at 100000g). Hence the reconstituted membrane contained about 25-fold more immunoglobulin than could be accounted for by trapping, even if the entire volume of the pellet consisted of unreconstituted protein solution. On the other hand, it has been demonstrated that proteins that are not known to be membrane constituents can bind to or interact with model membranes (Marinetti & Pettit, 1968; Weissman et al., 1974) and with plasma-membrane vesicles derived from Mycoplasma (Rottem et al., 1973). We have found that selectively reduced mouse serum IgM (in which all inter-chain disulphide bands have been cleaved and with a sedimentation coefficient of 7S in aqueous solvents) can adsorb to vesicles of pig mesenteric-node lymphocyte plasma membranes. The mouse IgM was added either to a cholate solution of the membrane before reconstitution, or to a suspension of membrane after reconstitution was completed. The reconstituted gradientisolated membranes contained about 0.09% and 0.04% (w/w) respectively of the mouse serum IgM (S. I. Chavin, A. Holliman, N. Richardson & A. Feinstein, unpublished work). These amounts are similar to the amount of pig membrane-associated immunoglobulin that is reincorporated in the conventional reconstitution experiments as described above. Hence, it is not possible to rule out adsorption of soluble immunoglobulin to the membrane vesicles in a way that is different from the attachment of the original membrane-associated immunoglobulins.

270

The reasons for the relatively low reincorporation of immunoglobulin are not known. Although the imunoglobulin clearly is associated with the purified membrane fraction, there is insufficient information about the immunoglobulin to say whether it is an integral or a peripheral membrane protein (Singer, 1974). The small fraction that is reincorporated needs to be examined for structural properties that are different from those of the bulk of the membrane-associated immunoglobulin and which might account for its preferential reassociation with membranes. We thank Dr. A. Feinstein for suggesting the reconstitution experiments and providing antisera. We acknowledge the collaboration of Dr. S. M. Johnson on some of the reconstitution experiments and thank Dr. F. B. P. Wooding and Dr. E. A. Munn for the electron-

microscopic studies.

References Allan, D. & Crumpton, M. J. (1970) Biochem. J. 120,133143 Changeux, J.-P., Huchet, M. & Cartaud, J. (1972) C. R. Hebd. S&ances Acad. Sci. Ser. D 274, 122-125.

S. I. CHAVIN AND A. HOLLIMAN Chavin, S. I., Johnson, S. M. & Holliman, A. (1975) Biochem. J. 148, 417-423 Crumpton, M. J. & Parkhouse, R. M. E. (1972) FEBS Lett. 22, 210-212 Heremans, J. F. (1971) Methods Immunol. Immunochem. 3,213-224 Kagawa, Y. (1972) Blochim. Biophys. Acta 265, 297-338 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Marinetti, G. V. & Pettit, D. (1968) Chem. Phys. Lipids 2, 17-34 Metcalfe, S. M., Metcalfe, J. C. & Engelman, D. M. (1971) Biochim. Biophys. Acta 241, 422-430 Neville, D. M., Jr. & Glossmann, H. (1971) J. Biol. Chem. 246,6335-6338 Racker, E. & Stoeckenius, W. (1974) J. Biol. Chem. 249, 662-663 Razin, S. (1972) Biochim. Biophys. Acta 265, 241-296 Rottem, S., Hasin, M. & Razin, S. (1973) Biochim. Biophys. Acta 298, 876-886 Singer, S. J. (1974) Adv. Immunol. 19, 1-66 Slack, J. R., Anderton, B. H. & Day, W. A. (1972) Biochim. Biophys. Acta 323, 547-559 Tillack, T. W., Carter, R. & Razin, S. (1970) Biochimn. Biophys. Acta 219, 123-130 Warren, G. B., Toon, P. A., Birdsall, N. J. M., Lee, A. G. & Metcalfe, J. C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71,622-626 Weissman, G., Brand, A. & Franklin, E. C. (1974) J. Clin. Invest. 53, 536-543

1975