The N-terminal heterogeneity of edta-extractable erythrocyte membrane proteins

Biochimiea et Biophysica Acta, 386 (1975) 107-119

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA36962 T H E N - T E R M I N A L H E T E R O G E N E I T Y OF E D T A - E X T R A C T A B L E ERYT H R O C Y T E M E M B R A N E PROTEINS

MICHAEL J. DUNN, WILLIAM MCBAY and ALUN H. MADDY Department of Zoology, Universityof Edinburgh, West Mains Road, Edinburgh EH9 3JT (U.K.)

(Received April 16th, 1974) (Revised manuscript received August 13th, 1974)

SUMMARY N-Terminal analysis of the proteins extracted from ox (Bos taurus) erythrocyte membranes by dilute EDTA is used as a means of estimating the heterogeneity of the protein fractions. Dinitrophenylation and dansylation reveal up to 8 different N-terminal amino acids in fractions which electrophoresis after dodecylsulphate treatment shows as having far fewer polypeptide chains. These fractions are prepared by gel electrophoresis in the presence and absence of detergent. Molecular weight estimation by the Ferguson procedure of the components revealed in the EDTA extract by electrophoresis in the absence of detergent confirms the large size concluded from electrophoresis with dodecylsulphate. The N-terminal analyses indicate that the high molecular weight of the complexes, both in the presence and absence of detergent, is more probably due to their being complexes of several polypeptides than exceptionally long single polypeptide chains.

INTRODUCTION Current knowledge of the polypeptide composition of membranes rests heavily on the results obtained from the analysis of membrane proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. We have previously questioned the assumptions that underlie the interpretation of some of these results [1-4], and also reported experimental data which indicate that protein aggregates, with an apparent molecular weight around 200 000 which cannot be dissociated by dodecylsulphate, can be formed from smaller units of 40 000 molecular weight [3, 4]. Whichever buffer system is used for electrophoresis, be it with or without detergent, it would be unwise to assume that the number of bands detectable within a gel after electrophoresis necessarily represents the number of protein species that make up the mixture. One band could consist of an aggregate of many proteins, or one protein could aggregate with itself in several ways to produce several bands. The complexities of the proteins are such that little confidence can be placed in the homogeneity of any fractions obtained by only one fractionation procedure, indeed a shadow of

108 doubt must remain over the homogeneity of any protein preparation until it has been extensively characterised e.g. by amino acid analysis and peptide mapping. As a first step in the characterisation of the fractions resulting from the gel electrophoresis of those erythrocyte membrane proteins extracted by dilute EDTA [1-10], we have identified their free N-terminal amino acids. The heterogeneity and identity of the N-terminal acids in the proteins of the ox cell membrane have already been briefly published by ourselves [3, 4] and the general result has been subsequently confirmed for the human cell by Knufermann et al. [11]. Such analysis reveals the minimum number of polypeptide chains in each fraction but the results are incompatible with the claims made for the proteins in the extract based on dissociation by dodecylsulphate and gel electrophoresis. It has been repeatedly concluded (e.g. refs 6 and 7) from such electrophoresis of an EDTA extract of erythrocyte membranes that it contains a complex of bands in the 200 000 molecular weight region (Peptides l and II by the nomenclature of Fairbanks et al. [6]) and a second protein with a molecular weight of about 40 000 (Peptide V). Because the presence of detergent is in many ways undesirable, we have also fractionated the mixture by polyacrylamide gel electrophoresis in a Tris/glycine buffer and related the two patterns. Briefly, bands of low mobility in Tris are composed of the high molecular weight polypeptides as defined by the dodecylsulphate method, and the bands of high mobility in Tris consist of the 40 000 protein of the dodecylsulphate pattern. We initially expected the various fractions, which were isolated by a preparative slab technique, to have different N-terminal amino acids, but they were identical, Peptides I + II being the same as Peptide II alone and the same as Peptide V. To check these results obtained on the fractions isolated in a Tris buffer, we isolated Peptides 1 and lI separately by the electrophoresis of whole ghosts in a gel slab with dodecylsulphate and again found the same set of N-terminal amino acids. The significance of these results is discussed. METHODS

(1) Ethylenediamine tetra-acetic acid (EDTA) extraction Ghosts were prepared from ox (Bos taurus) blood as previously described [12] by modification of the procedure of Dodge et al. [13]. The ghosts, diluted with four volumes of 0.5 mM EDTA, adjusted to pH 7.5 with NaOH, were left to extract for 2 h at room temperature and the insoluble residue removed by centrifugation at 65000 x g f o r l h.

(2) Polyacrylamide gel electrophoresis Analytical polyacrylamide gel electrophoresis was carried out using either a Tris/glycine/EDTA buffer, the Tris system (a 5 9/o acrylamide/0.1 ~ bisacrylamide gel in a 5 mM Tris/glycine buffer containing 0.5 mM EDTA adjusted to pH 8.0 [9]), or a dodecylsulphate containing phosphate buffer, the sodium dodecylsulphate system, ( 5 ~ acrylamide/0.135~ bisacrylamide [14]). Samples were prepared for electrophoresis in the sodium dodecylsulphate system by addition of dithioerythritol to a final concentration of 20 mM, made 59/o with sodium dodecylsulphate, heated at 100 °C for 3 min, and finally made 8 M with respect to urea. For the Ferguson analysis [15] the conditions of the Tris system were kept constant except for the

109 acrylamide concentration which ranged from 3.5 to 9 . 0 ~ and the mobility of the proteins measured relative to bromphenol blue. Preparative gel electrophoresis was carried out by a vertical slab method using either buffer system. During the prerunning and running procedures the buffers in the two electrode chambers were continuously recirculated by a peristaltic pump. In both systems 130 ml of the appropriate acrylamide solution were [9, 14] polymerised in a mould 20 × 20 × 0.4 cm. For the Tris system the slab was prerun for 6 h at 7 V.cm -1 and then 5 ml of EDTA extract protein solution (5 mg/ml) were applied and electrophoresed for 18 h at 6 V.cm-1. In the case of the sodium dodecylsulphate system which was used to prepare the dodecylsulphate derived fractions, Peptides I and II, the slab was prerun for 18 h at 4 V.cm -1. 3 ml of ghost suspension (5 mg/ml) was heated at 100 °C in the presence of 5 ~ dodecylsulphate and 20 mM dithioerythritol for 3 min, and then made 8 M urea. This solution was applied to the slab and electrophoresed at 4 V.cm-1 for 72 h. After electrophoresis the slabs were cut up into 0.5-cm slices and each slice homogenised in 10 ml of either 2.5 mM phosphate buffer + 0.5 mM EDTA (pH 8.0) lbr Tris slabs, or 5 mM NaHCO3 containing 0.05 ~o dodecylsulphate for the detergent slabs. The homogenates were left overnight at 4 °C, spun, re-extracted with the same buffer and the supernatants from each homogenised slice pooled.

(3) Determination of N-terminus (a) Fluorodinitrobenzene (N~ph-F). 10 ml of extract (5 mg protein/ml) were dinitrophenylated overnight at room temperature under Sanger's conditions [16] and the protein then precipitated in 5 ~ trichloroacetic acid. The precipitate was washed with ethanol and water before hydrolysis in 5.7 M HC1 for 16 h at 105 °C. The dinitrophenylated N-terminal acids were extracted into ether, the ether evaporated off and the dinitrophenol derivatives dissolved in 2 ~ acetic acid. Dinitrophenol was then removed by passage through an alumina column [17]. The dinitrophenol-amino acids were identified by two-dimensional thin-layer chromatography on silica plates (polygram-SIL.N-HR, Camlab) using two pairs of solvents, benzene/acetic acid (9:1, v/v) + chloroform/methanol/acetic acid (95: 5:1, by vol.) and butanol/ammonia (n-butanol saturated with 0.1 ~ ammonia) + chloroform/methanol/acetic acid (95:5:1, by vol.). Reference dinitrophenol-amino acids were obtained from B.D.H. Ltd. (b) Dansyl chloride (DNS-Cl). 0.5-ml samples of EDTA extract, Tris slab fractions, or sodium dodecylsulphate slab fractions were used. In order to react in small volumes each sample was split into 5 x 0.1-ml aliquots and each made 0.3 M NaHCO3, 8 M urea. 0.1 ml Dns. C1 (5 mg/ml) in acetone was added to each aliquot and the aliquots reacted overnight in the dark at room temperature in tubes sealed with parafilm. The five aliquots of each sample were pooled and precipitated in icecold 5 ~ trichloroacetic acid. The precipitates were spun, washed twice with 1 M HC1, and hydrolysed with 5.7 M HC1 for 18 h at 105 °C [18]. The hydrolysates were dried in vacuo, redissolved in 100 #l 5 0 ~ aqueous pyridine and spotted onto small pieces of filter paper, dried and eluted with 100/A water-saturated ethyl acetate. (By this procedure the bulk of the D n s . O H and Dns.NHz, which would otherwise interfere with the subsequent chromatography, were removed from the dansylated amino acids [19]). The ethyl acetate extracts were dried in vacuo and redissolved in 5 0 ~ aqueous pyridine for application to the thin-layer plates. Pclyamide thin-layer plates

110 (B.D.H. Ltd) were used, developed with n-heptane/n-butanol/glacial acetic acid (3:3:1, by vol.) in the first dimension, and toluene/glacial acetic acid (9:1, v/v) in the second dimension [20]. Reference Dns.amino acids were obtained from B.D.H. Ltd. RESULTS In order to minimise proteolytic and other damage, we, and others [6], have shortened the time of extraction from the prolonged procedure previously used [5, 8]. 2 5 - 3 0 ~ of the membrane protein is released into 0.5 mM EDTA after 2 h at 20 °C and a further 10~o by subsequent dialysis for 16 h at 4 °C. About 20~o is released if the initial extraction is performed at 4 °C. These different extracts do not differ in their N-terminal contents or gel electrophoretic patterns. Our routine procedure is chosen as a convenient way of preparing a relatively concentrated protein solution. The many similarities between the proteins extracted from the ox membrane and those from other species (electrophoretic behaviour, sedimentation characteristics, virtual absence of carbohydrate and lipid, and fibrous nature) have been discussed previously [8]. The careful removal of leucocytes, the low ionic strength, the presence of EDTA during extraction, and reduction, heating and urea treatment prior to electrophoresis with dodecylsulphate all serve to minimise proteolysis. There is, to our knowledge, no previous report of proteolytic activity in the EDTA extracts of erythrocyte membranes and we cannot detect any activity when using casein, haemoglobin, azo-albumin (Sigma Chemical Co. Ltd) or Azocoll (Calbiochem Ltd) as test substrates. The result of electrophoresing the EDTA extract in the sodium dodecylsulphate system is seen in Fig. 1. The pattern of the ox extract is essentially the same as that obtained from the corresponding human extract, with protein in the 200 000 and 40 000 molecular weight regions. The 200 000 tool. wt complex has been described as a doublet (Peptides I and II) and although for convenience we shall refer to it as a doublet it should be stressed that, as has previously been reported by Lenard [14], up to five bands may be discerned in this region. (Peptides I and II both subdivide and other narrow bands are observed. This heterogeneity is difficult to demonstrate photographically but is illustrated in the densitometric trace, Fig. 2.). Of the pair of bands in the 40 000 mol. wt region, the smaller (actual value 45 000 mol. wt) predominates in the ox and probably corresponds to a protein of similar size from human membrane (Peptide V) but the second band (50 000 mol. wt) is relatively stronger in ox. In addition to these major bands a sharp band or complex of bands of very low mobility is consistently observed together with many trace bands between the 200 000 and 40 000 molecular weight regions. The fractionation of the EDTA extract by polyacrylamide gel electrophoresis in the Tris system is shown in Fig. 1. There are three major groups of bands of low mobility a, fl, and 7 and also protein of very high mobility o.~. The relationship between the patterns produced by the two buffer systems, illustrated in Figs I and 3, was established by adapting the Tris system to a preparative scale using a vex tical slab gel and after electrophoresis slicing the slab and analysing the proteins in each slice by both electrophoretic systems. The pooled slices fall into five categories, Fractions ct,/3, 7, e and (.. In order to exclude any insoluble, unfractionated prGtein the topmost slice of the gel is not pooled. It does not usually contain

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Fig. 1. The examination of the fractions of the EDTA extract of ox erythrocyte ghosts isolated by preparative polyacrylamide gel electrophoresis in the Tris system. In the upper series each slice of the preparative slab is examined in the analytical Tris system and in the lower series these same fractions, some having been pooled as indicated, are examined in the analytical sodium dodecylsulphate system. In the series from the slab the gel to the left is taken from the top of the slab, i.e. the - v e pole, and the gels to the right from progressively further down the slab. Gels stained in Coomassie blue.

much protein and that present consists o f a mixture o f Fractions a and fl (Fig. 1). By examining Fig. 3 it will be seen that a and fl both contain Peptides I and II o f the sodium dodecylsulphate system: in the Tris system a contains the a band, and fl the fl band. Fraction ), in sodium dodecylsulphate contains only Peptide II and in Tris, Band 7. ()' could be the faster c o m p o n e n t o f Peptide II ( k n o w n as II-1) but the slight difference in mobility that distinguishes II-1 is not sufficient to draw this conclusion with any confidence), e is made up o f the proteins of intermediate mobility in Tris and is highly heterogeneous in sodium dodecylsulphate, while o contains the proteins o f high mobility (i.e. 40 000 mol. wt in sodium dodecylsulphate in both buffers. These results were amplified and confirmed by examination o f Bands a, fl and 2" in the Tris system by the Ferguson procedure, i.e. measurement of the effect o f polyacrylamide concentration on mobility. The slope of the mobility versus polyacrylamide concentration for y is greater than that of aldolase (mol. wt 160 000) but less than fibrinogen (mol. wt 310 000) i.e. is consistent with a molecular weight e f 200 000-250 000. The m e t h o d indicates that protein o f band fl is bigger than fibrinogen as would be expected o f a complex of 200 000 mol. wt chains (i.e. Peptides I + II).

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Fig. 2. A densitometer trace of the electropherogram of ox ghosts fractionated in the sodium dodecylsulphate system. 0, indicates where a Coomassie blue staining band is apparent by visual inspection of the gel.

The possibility of the intimate association of Peptides I and II within the intact membrane has previously been indicated by studies using cross-linking agents [21] and Band fl might therefore represent the state of the proteins in the membrane. The Ferguson plot shows that components of Band a are even bigger than those of fl and the probability that a is an aggregate of fl is supported by the identical mobilities (as calculated by extrapolation) of the two fractions at zero gel concentration. Band ~ cannot be analysed by this method because as gel concentration is increased the band becomes progressively more diffuse. N-Termini of the unfractionated EDTA extract were identified by dinitrophenylation and dansylation. Both methods revealed tyrosine, lysine, glycine, threonine, serine, aspartic and glutamic acids. Dansylation also detected alanine, leucine or valine (this pair is not separated on the chromatograms) and ethanolamine. As serine and ethanolamine were shown by dansylation to be present in lipid extracts Gf the membrane, the presence of ethanolamine, at least, in the extract probably indicates a trace contamination by lipid, but the serine could also arise from a polypeptide chain. Only very low levels of lipid phosphate were detectable in the EDTA extract. Passage of the extract through a Sephadex G-200 column in a dilute phosphate buffer did not affect the N-terminal composition of the mixture. The small quantities of Fractions a, fl, 7, e and o~ derived from polyacrylamide gels only permitted analysis by the more sensitive dansylation method which produced the unexpected result that all fractions contained the same N-terminal amino acids, including the serine and ethanolamine. Identity of c~ and fl was to be anticipated but the same result for 7 was surprising, and the further identity of e and ~ could not

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Fig. 3. A diagrammatic representation of the results shown in Fig. 1. The result obtained when the complete E D T A extract is fractionated in the sodium dodecylsulphate system is shown on the left, and in the Tris system on the right. The small diagrams illustrate the analysis of a, fl, y and co in the sodium dodecylsulphate system after their isolation by the fractionation of the complete extract in the Tris system run on a preparative scale.

reasonably be ascribed to chance. In order to eliminate the possibility that polyacrylamide, or any other contaminant, was responsible for any of the N-termini, a slab was run in the absence of a protein sample and the resulting fractions analysed by the dansylation procedure. N o N-terminal amino acids were found. When E D T A extract containing added haemoglobin was run on a slab and the fractions containing

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Fig. 4. The mobility of the major protein bands (a, fl, 7) of the EDTA extract in the Tris system as a .function of acrylamide concentration. Mobility is expressed relative to bromphenol blue. The curves for aldolase and fibrinogen are shown ( . . . . . . . ). The slopes are calculated by the method of least squares. Bisacrylamide is kept at 0.1 ~ .

haemoglobin were analysed for N-terminus, an additional spot, methionine, and an enhanced spot, valine, were observed in addition to fainter traces of the spots normally seen in slab fractions from EDTA extract. Haemoglobin when analysed alone was found to contain only N-terminal valine and methionine, i.e. the previously reported N-termini[22]. To further insure and check that the N-terminal data relating to the proteins derived from the Tris system could not be a consequence of some unforseen anomaly these same proteins, i.e. Peptides I and II, were prepared directly from ghosts by electrophoresis of dodecylsulphate solubilised ghosts in the sodium dodecylsulphate system. In these detergent derived preparations of pure Peptide I and pure Peptide 11 the same pattern of N-termini was again obtained, including ethanolamine. The presence of ethanolamine suggests incomplete separation of lipid from protein even after detergent treatment. The quantity of N-terminal groups, while cogent, is difficult to measure, the percentage recovery from acid hydrolysis of a complex mixture cannot readily be measured, but using data from the hydrolysis of mixtures of the relevant dinitrophenol-

115 amino acids we estimate 1 mol of dinitrophenol -amino acid per 60 000-80 000 g protein of the unfractionated EDTA extract. The result must be accepted with caution but it does indicate significant amounts of N-termini. The dansyl method is not readily quantified, and quantitative analysis of the small amounts of protein eluted from gels is not possible. However, no differences can be detected between the fractions by visual examination of the thin-layer plates under ultraviolet illumination. DISCUSSION The results indicate that the protein bands known as Peptides 1, II and V (it is not strictly correct to refer to Peptide V as one band as it always contains two, 45 000 and 50 000 mol. wt) contain a minimum of eight polypeptides with free N-termini (different chains may have identical ends, there may be other chains with blocked termini and also a protein with N-terminal serine) and these free ends are the same in Peptides I, II and V. Our original report [3] of the N-terminal heterogeneity of the "large polypeptides" of the erythrocyte membrane has been confirmed and extended using human cells [11, 24] and the same N-termini observed in different bands of the dodecylsulphate-containing gels. Although amino acid analysis of what are probably heterogeneous protein samples has only limited significance, the compositional identity of the fractions again points to their containing the same polypeptides (in preparation). It should also be noted that the EDTA extracts of human [24, 25] and ox [26] cells are immunologically heterogeneous and Fractions a, fl, 7, e and oJ of the ox cell contain many common antigens. A close relationship between proteins in Fraction to and those in a, /3 and possibly 7 is still further supported by our earlier observation [3, 4] that passage of ~o in the absence of detergent, through a Sephadex column results in the formation of aggregates in the 200 000 molecular weight range. While we do not readily subscribe to the frequently invoked notion of "proteolysis" as an explanation of every anomalous result obtained by electrophoresis with dodecylsulphate, proteolysis simply cannot be produced as a plausible explanation of our results. The analyses were confirmed on the high molecular weight (as defined by the detergent method) fractions of ghosts which prior to electrophoresis had been reduced and heated for 5 min at 100 °C in 5 ~o dodecylsulphate. Proteolytic activity cannot be detected in the EDTA extract. The following alternative explanations need to be considered: (a) The N-terminal identity of the fractions is fortuitous and each happens to contain several chains of the appropriate molecular weight i.e. 250 000 or 205 000 or 45 000-50 000. This explanation would be very difficult to disprove, but would represent a remarkable coincidence. (b) Each of the fractions has been contaminated by a set of peptides and the termini of these peptides obscure any terminal differences between the major protein constituents of each fraction. If dodecylsulphate has the dissociative ability usually ascribed to it, it is difficult to reconcile this explanation with the results obtained on the "large" molecular weight fractions eluted from dodecylsulphate-containing gels. (c) The N-termini of the different fractions are identical because each fraction consists of non-covalently linked aggregates constituted of various combinations of a small set of polypeptide chains. This explanation would be readily adopted but for

116 (i) the fairly widely held belief that the EDTA extract of erythrocytes consists predominantly of one protein, spectrin, and (ii) the even more widely held belief that after reduction of any disulphide bonds dodecylsulphate dissociates all protein aggregates into their component polypeptides which can then be electrophoretically separated in the order of their molecular weights. (i) The relative homogeneity of the EDTA extract was initially claimed by Marchesi and his co-workers [5, 23] on two grounds. (a) Electrophoresis in the presence of dodecylsulphate indicated one component (occasionally two were noted) with a molecular weight of 140 000. Values ranging from 92 300 to 156 000 tool. wt were obtained by centrifugation but the values varied with rotor speed. (b) On the basis of its methionine content and a molecular weight of 140 000 the number of CNBr cleavage products of the EDTA extract was consistent with its being one polypeptide chain. In the first place the results of the electrophoresis are contrary to those of several other workers [6-9]. Not only are several bands detectable within the gels, but their molecular weights are quite different, 200 000-250 000 and, according to most authors, a small amount of protein around 40 000. Secondly, as the CNBr peptides were also analysed by the sodium dodecylsulphate system, only those fixed by trichloroacetic acid would have been observed, and perhaps more seriously, the agreement between the number predicted and the number found is only significant for a chain of 140 000 daltons. The additional claim of immunological homogeneity [23] of the extract has been challenged by others using more sensitive techniques, either fused rocket for human extracts [25] or the Oakley-Fulthorpe method and modified Ouchterlony method for ox proteins [26]. (ii) Gel electrophoresis in the presence of dodecylsulphate has been so extensively used for the analysis of membrane proteins that the validity of the conclusions based on the method cannot be lightly set aside. The technique has a central role in membrane protein technology and has the advantage of producing consistent results, but it is doubtful that the theoretical foundations of the method are adequate to bear the enormous superstructure that has been placed upon them and certainly insufficient to assume its universal applicability. Three assumptions underlie the conclusions drawn from this technique. (1) After cleavage of the disulphide bonds all complexes are dissociated into their constituent polypeptide chains. (2) All polypeptides, bv binding the same amount of detergent per unit chain length, are transformed into complexes of identical conformation which differ only in one dimension i.e. length. (3) The electrophoretic mobility of these complexes when measured under suitable conditions is a simple logarithmic function of molecular weight. (1) The first assumption has the most direct relevance to the interpretation c f data presented in this paper. The evidence on which it is based must come from the behaviour of proteins whose polypeptide chain composition is known and these are, almost exclusively, soluble proteins. The extrapolation of such data to unknown. insoluble, proteins therefore implies that the interactions of the insoluble proteins are no more intractable than those of soluble proteins. But it is the very intractability of the associations of membrane proteins with themselves or lipids that makes their study so difficult, and to ignore this difference is to tacitly beg the entire question. This theoretical objection is supported by several reports of membrane protein aggregates that are not dissociated by standard dodecylsulphate treatments. Organic sol-

117 vents have been found by two laboratories [6, 27] to cause the association of some membrane proteins into aggregates which are not dissociated by the detergent, and similarly resistant complexes can be caused by Ca 2+ [28]. Many published photographs of the electrophoresis of membrane proteins with dodecylsulphate show protein at the gel origin but the logically necessary conclusion that this material of infinitely low mobility is infinitely long has not been drawn. Aggregation and disaggregation of human erythrocyte glycoproteins in the presence of the detergent has recently been reported [29]. (2) Compared with the number of proteins known to exist, the number of proteins for which dodecylsulphate binding characteristics have actually been measured is vanishingly small, and even amongst this small number several anomalies have already been reported. Thus in a study of only 19 proteins [30] dodecylsulphate binding varied from 0.2 to 2.2 mg per mg protein, the binding of the detergent by virus coat proteins [31] ranges from 0.8 to 2.2 mg detergent per mg protein and the binding by amylase and its apparent molecular weight are totally anomalous [32]. Furthermore a constant binding ratio per unit polypeptide chain length for all proteins requires binding to be independent of amino acid composition and this is not the case. Acidic proteins bind a disproportionally small amount of detergent and basic proteins a correspondingly large amount [30]. (3) There is extensive data in favour of the relationship between mobility and molecular weight although anomalies have been reported, e.g. the effects of maleylation [33], however the data are not valid if the detergent does not dissociate all polypeptide complexes. The confirmation of the size distribution of the polypeptides of the erythrocyte membrane as determined by electrophoresis in the presence of dodecylsulphate has been persuasively supported by gel exclusion chromatography on agarose in the presence of guanidine. HC1 [34, 35]. The limitations of this latter method are clearly stated by its authors and, in addition, in the case of membrane proteins, certain interactions not dissociated by guanidine. HC1 have been reported [4]. (The stability of agarose beads in 6 M guanidine. HC1 exemplifies one type of non-covalent association resistant to the salt). The behaviour of the 200 000 mol. wt complexes in guanidine on the agarose columns [34, 35] argues strongly against their being globular bodies of 200 000 mol. wt, but is less conclusive for a highly asymmetric complex, and the authors of the method specifically noted the difficulty of predicting the behaviour of a branched molecule on their columns. (d) A fourth explanation of our findings is that the high molecular weight complexes are covalently linked forms of the 40 000 mol. wt sub-units. Our findings could be readily explained by the presence in the EDTA extract of cross-linked complexes of relatively small polypeptides, the 200 000 mol. wt complexes, together with the free subunits, 40 000 mol. wt chains. The true size of the complexes is not known as neither electrophoresis with dodecylsulphate or gel exclusion chromatography with guanidine.HC1 can be used to provide a valid molecular weight determination of cross-linked molecules. This would have been, until quite recently, little more than a formal explanation of our results but it has now been strongly supported by the reports of e-lysyl-7-glutamyl peptide bonds [36] and other lysine derived bonds [37] between proteins of plasma membranes. On balance, therefore, at our present state of knowledge the presence of

118 b r a n c h e d complexes o f covalently linked p o l y p e p t i d e s in the E D T A extract o f the m e m b r a n e is p e r h a p s the most likely e x p l a n a t i o n o f o u r findings. However, the t h i r d possibility i.e. o f n o n - c o v a l e n t associations c a n n o t be excluded a n d gains some supp o r t f r o m our previous o b s e r v a t i o n s [3, 4] t h a t passage o f F r a c t i o n s e or ~ d o w n a Sephadex G-200 c o l u m n results in the f o r m a t i o n o f a d o u b l e t in the 200 000 m o l e c u l a r weight region on electrophoresis with d o d e c y l s u l p h a t e : it w o u l d be surprising if this p r o c e d u r e were to induce the f o r m a t i o n o f covalent b o n d s between p o l y p e p t i d e chains. (We m u s t a d d t h a t there is no evidence which unequivocally excludes any o f the f o u r ( a - d ) possible e x p l a n a t i o n s or t h a t a c o m b i n a t i o n o f factors is operative). W h a t e v e r the m o l e c u l a r basis for our present results they indicate t h a t the 200 000 a n d 40 000 mol. wt complexes c o n t a i n m a n y p o l y p e p t i d e chains a n d also i m p l y a close similarity between the two, a n d the n o t i o n o f their consisting o f the same p o l y p e p t i d e chains in differing states o f aggregation is s u p p o r t e d by our earlier o b s e r v a t i o n of their interconvertability. The interactions o f m e m b r a n e p r o t e i n s are p r o b a b l y therefore, c o n s i d e r a b l y more c o m p l e x t h a n has been previously assumed. T h e biological significance o f these associations between proteins, or their m o l e c u l a r basis, r e m a i n s unexplained. If, as has frequently been suggested, the extrinsic p r o t e i n s affect the mechanical p r o p e r t i e s o f m e m b r a n e s , the t r a n s i t i o n between the 200 000 a n d 40 000 m o l e c u l a r weight states could be one aspect o f the control o f the m e m b r a n e ' s rigidity and m o b i l i t y and m a y m e d i a t e the effect o f drugs such as cytochalasin B on the m o b i l i t y o f m e m b r a n e components. ACKNOWLEDGEMENT We are indebted to the M e d i c a l Research Council for financial support. REFERENCES 1 Maddy, A. H. (1971) in The Red Cell Membrane (Weed, R. I., Jaffe, E. R. and Miescher, P. A., eds), pp. 27-47, Grune and Stratton, New York 2 Maddy, A. H. (1972) Sub-Cell Biochem. 1,293-301 3 Maddy, A. H. and Dunn, M. J. (1973) in Protides of the Biological Fluids (Peeters, H., ed.), Vol. 21, p. 21-27, Pergamon, Oxford 4 Dunn, M. J. and Maddy, A. H. (1973) FEBS Lett. 36, 79-82 5 Marchesi, S. L., Steers, E., Marchesi, V. T. and Tillack, T. W. (1970) Biochemistry 9, 50-57 6 Fairbanks, G., Steck, T. L. and Wallach, D. F. H. (1971) Biochemistry 10, 2606-2616 7 Hoogeveen, J. Th., Juliano, R., Coleman, R. and Rothstein, A. (1970) J. Membrane Biol. 3, 156-172 8 Maddy, A. H. and Kelly, P. G. (1971) Biochim. Biophys. Acta 241, 290--301 9 Maddy, A. H., Dunn, M. J. and Kelly, P. G. (1972) Biochim. Biophys. Acta 288, 263-276 10 Steck, T. L. and Yu, J. (1973) J. Supramol. Struct 1,220-232 11 Knufermann, H., Bhakdi, S., Schmidt-Ullrich, R. and Wallach, D. F. H. (1973) Biochim. Biophys. Acta 330, 356-361 12 Maddy, A. H. (1966) Biochim. Biophys. Acta 117, 193-200 13 Dodge, J. T., Mitchell, C. and Hanahan, D. J. (1963) Arch. Biochem. Biophys. 100, 119-130 14 Lenard, J. (1970) Biochemistry 9, 1129-1132 15 Rodbard, D. and Chrambach, A. (1971) Anal. Biochem. 40, 95-134 16 Sanger, F. (1945) Biochem. J. 39, 507-515 17 Turba, F. and Gundlach, G. (1955) Biochem. Z. 326, 322-324 18 Gray, W. R. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 139-151

119 19 Seiler, N. (1970) in Methods in Biochemical Analysis (Glick, D., ed.), Vol. 18, pp. 259-338, Interscience, New York 20 Woods, K. R. and Wang, K. T. (1967) Biochim. Biophys. Acta 133, 369-370 21 Hulla, F. W. and Gratzer, W. B. (1972) FEBS Lett. 25, 275-278 22 Ozawa, H. and Satake, K. (1955) J. Biochem. Tokyo, 42, 641-648 23 Tillack, T. W., Marchesi, S. L., Marchesi, V. T. and Steers, E. (1970) Biochim. Biophys. Acta 200, 125-131 24 Langdon, R. C. (1974) Biochim. Biophys. Acta 342, 213-228 25 B~g-Hansen, T. C. and Bjerrum, O. J. (1973) in Protides of the Biological Fluids (Peeters, H., ed.), Vol. 21, pp. 3945, Pergamon, Oxford 26 Green, J. R., Dunn, M. J., Spooner, R. L. and Maddy, A. H., (1974) Biochim. Biophys. Acta, 373, 51-57 27 Tanner, M. J. A. and Boxer, D. H. (1972) Biochem. J. 129, 333-347 28 Triplett, R. B., Wingate, J. M. and Carraway, K. L. (1972) Biochem. Biophys. Res. Commun. 49, 1014-1020 29 Azuma, J., Janado, M. and Onodera, K. (1973) J. Biochem. Tokyo 73, 1127-1130 30 Nelson, C. A. (1971) J. Biol. Chem. 246, 3895-3901 31 Simons, K. and Kaariainen, L. (1970) Biochem. Biophys. Res. Commun. 38, 981 32 Mitchell, E. D., Riquetti, P., Lording, R. H. and Carraway, K. L. (1973) Biochim. Biophys. Acta 295, 314-322 33 Tung, J. S. and Knight, C. A. (1971) Biochem. Biophys. Res. Commun. 42, 1117-1121 34 Gwynne, J. T. and Tanford, C. (1970) J. Biol. Chem. 245, 3269-3271 35 Trayer, H. K., Nozaki, Y., Reynolds, J. A. and Tanford, C. (1971) J. Biol. Chem. 246, 4485-4488 36 Birckbichler, P. J., Dowben, R. M., Matacic, S. and Loewy, A. C. (1973) Biochim. Biophys. Acta 291, 149-155 37 Langdon, R. G. (1974) Biochim. Biophys. Acta 342, 229-236