Proteins and glycoproteins of the erythrocyte membrane

ARCHIVES

OF

BIOCHEMISTRY

Proteins

AND

and

DAVID

BIOPHYSICS

475487 (1972)

148,

Glycoproteins

KOBYLKA,

of the Erythrocyte

ARUN KHETTRY, BAK KERMIT L. CARRAWAY

Department of Biochemistry,

Oklahoma

Membrane’

C. SHIN,

AND

State University, Stillwater, Oklahoma 74074

Received September 21, 1971; accepted October 27, 1971 Erythrocyte membranes from several species were prepared by three different methods of hypotonic hemolysis and examined for variations in protein and glycoprotein content by acrylamide gel electrophoresis in sodium dodecyl sulfate. Significant variations were noted in morphology of the membranes prepared by the different methods without attendant variations in protein patterns of the major membrane proteins for most cases observed, which show a similar pattern of nine common bands for all of the species observed. The significant difference in protein pattern which was noted was attributed to proteolytic digestion of membranes which were fragmented during preparation. Failure to remove white blood cells from membrane preparations was shown to be a significant source of the problem with proteolytic digestion. Glycoproteins were analyzed by a&amide gel electrophoresis or by column chromatography. Each species appears to have a different major glycoprotein (or group of closely related glycoproteins). Molecular weights of glycoproteins calculated from acrylamide gel electrophoresis were found to vary with the percentage of acrylamide in the gel, indicating that these proteins do not behave in a normal fashion in this electrophoresis system. The molecular weight calculated from gel filtration data for the human membrane glycoproteins (26,000) was quite disparate from those calculated from gel electrophoresis (88,000 to 62,000 in 5 to 10% gels).

A knowledge of the distribution of proteins (or polypeptide chains) of cellular membranes is essential to the development of concepts of membrane structure. Erythrocyte membranes offer a well-defined source of material for studying membrane proteins. Comparative studies between species are of interest because of the large variations in functional specificities that occur in the cells of different species. Examinations of proteins and glycoproteins from red cells of several species have resulted in conflicting reports concerning the similarities among species (l-3). The first reports of protein comparisons by acrylamide electrophoresis indicated differences between species that might be related to phylogenetic differences (1,2). More recently Lenard (3) has reported that almost all proteins in a group of five species appear 1 Publication No. 2359 of the Oklahoma Agricultural Experiment Station.

identical, with only a limited number of species specific bands. A major glycoprotein of mol wt 108,000 was reported by Lenard (3) which was not observed in the work of Carraway and Kobylka (2). Lenard (3) has also noted that dog membranes prepared by two different procedures showed quite different protein patterns. This suggests that variations in membrane preparative procedures may yield membranes with widely differing protein compositions. However, the source of these protein variations has not been specified. The current studies were undertaken to investigate possible sources of the changes in protein composition and to relate these to alterations in membrane morphology, if possible. Further studies of the glycoprotein components of erythrocyte membranes were also undertaken because of the ambiguities concerning their distribution and molecular weights. 475

Copyright

@ 1972 by Academic

Press,

Inc.

476

KOBYLKA MATERIALS

AND METHODS

Mate&Is. Chemicals for electrophoresis were obtained from Eastman (highest purity grade) or Canalco. SDS2 was a product of Sigma. The molecular weight markers for SDS electrophoresis calibration were myosin3, fl-galactosidase, bovine serum albumin, eatalase, ovalbumin, lactic dehydrogenase, chymotrypsinogen and cytochrome c (all products of Sigma). Membrane preparation. Human blood was obtained from the Dallas Community Blood Bank and used within 1 week of the withdrawal date. Animal blood samples were collected by the staff of the College of Veterinary Medicine, Oklahoma State University, in citrate or acid-citrate-dextrose solution. These samples were used within l-2 days of collection. The erythrocyte membranes for comparative studies were prepared by three different procedures: the hypotonic phosphate method of Dodge et al. (4), the Cat+ -Verona1 hypotonic lysis of Burger et al. (5), and the Tris-EDTA method of Marchesi and Palade (6). Bovine and human erythrocyte membranes for additional protein studies were prepared by the Dodge et al., procedure (4). Membranes were examined for morphological changes’ using a Leitz phase contrast microscope. Polyacrylamide gel electrophoresis. Gels were prepared and run according to standard procedures (7, 8). A fixed ratio of acrylamide to bisacrylamide was maintained for preparation of gels with different percentages of acrylamide. Samples were solubilized in 0.1 M phosphate buffer (pH 7.8) containing 2-3yo SDS and 1% mercaptoethanol. Solubilization could be acheived either by incubating at room temperature for 18 hr or by heating at 100° for 3 min (3). After each heat step, however, additional mercaptoethanol was added and the sample was incubated 5-18 hr at room temperature. Solubilisation and electrophoresis have been performed on lyophilized, freeze-thawed and fresh membrane samples with no discernible differences in patterns. Lyophilization causes decreased solubility of some treated membrane samples, which results in deposition of a large amount of material at the top of the gel during electrophoresis. Protein concentrations of membrane samples were determined by the procedure of Lowry et al. (9). The amount of protein applied to gels was varied depending on the length of gel used, 70-200 pg being applied to 6-10 cm gels and 100-350 pg applied to 13-15 cm gels. Protein was detected by staining with coomas2 Abbreviations used are: SDS, sodium dodecyl sulfate; PAS, periodic acid-Schiff procedure for carbohydrate staining; CB, coomassie blue. 3 Prepared by Mr. C. Schwartz.

ET AL. sie blue using three different procedures. (1) Gels were stained in 0.05ojneoomassie blue in 7yo acetic acid overnight and destained in 7% acetic acid, (2) gels were fixed l-2 hr in 10% TCA, rinsed and stained as in procedure 1, and (3) gels were stained in 0.05% coomassie blue, 9% methanol and 7% acetic acid overnight and destained in 7% acetic acid (8). Carbohydrate was detected by modifications of the periodate-Schiff method. It is essential to remove SDS from the gels before treatment. This was accomplished by stirring in a Hoefer destainer against either 40% methanol-70/, acetic acid’ or 15yo acetic acid (10) overnight. The gels are then treated with 1% periodic acid in 7yo acetic acid for 1 hr at room temperature in the dark and washed with 7% acetic acid to remove periodate. After a 1-hr treatment with Schiff reagent (11) at room temperature in the dark, the gels were washed 3-4 times with 1% sodium metabisulfite in 0.1 N HCl for 10 min each. In $1 examples shown gel scans were obtained with a Gilford 2000 spectrophotometer equipped with Model 2410 linear transport accessory, scanning at 550 nm for coomassie blue and PAS. The standard proteins which were used for estimation of molecular weights are given below with the molecular weight value used: 5% gels-myosin, 212,000 (12), p-galactosidase, 130,000 (S), catalase, 60,000 (8), and lactic dehydrogenase, 35,000 (12); 6% gels-@-galactosidase, bovine serum albumin, 66,500 (13), catalase, lactic dehydrogenase and chymotrypsinogen, 25,700 (8); 8% gels-bovine serum albumin, catalase, lactic dehydtogenase and cytochrome C, 11,700 (8); 10% gels-same as 8%. Samples were solubilized under the same conditions used for the membranes. Column chromatography. Solubilized membranes were chromatographed on Sepharose 4B in 1% SDS, 0.02% sodium azide and0.0 5 M phosphate (pH 7.0) on a 2.5 X 90-cm column. Fractions were monitored for absorbance at 280 nm and for carbohydrate by the DuBois procedure (14). Combined fractions from the peak areas were dialyzed against 40$Zomethanol to remove SDS and lyophilized. Samples were prepared for electrophoresis as above and for amino acid analysis by hydrolysis in 6 N hydrochloric acid at 110” for 22 hr (15). The standard proteins used to characterize the column for molecular weight estimations were myosin, &galactosidase, bovine serum albumin, ovalbumin, 43,000 (8) and cytochrome c. Neuraminidase treatment. Samples of washed human erythrocytes or ghosts were incubated with Clostridium perfringens neuraminidase (Sigma Type VI) according to the procedure of Eylar et * This procedure was kindly communicated to us by Dr. H. Glossman.

ERYTHROCYTE

MEMBRANE

al. (16). Separate samples of ghosts were hydrolyzed for 1.0 hr at, 80” in 0.1 N H&04. Supernatant sampIes were assayed for free sialic acid by the procedure of Warren (17). Ghosts were prepared from the erythrocytes by the procedure of Dodge, et al. (4), and both treated and control (no neuraminidase) samples were subjected to electrophoresis as described above. RESULTS

Membrane preparation. The variations noted in reports from different laboratories on the protein distributions of erythrocyte membranes could arise from variations in either the preparative techniques or the analytical methods. Therefore a comparison was made of the responses of the membranes of several different species to variations in the membrane isolation conditions. Three different isolation methods were used, the phosphate procedure of Dodge et al. (4)) the TrisEDTA method of Marchesi and Palade (6) and the Ca2+-veronal of method of Burger et al. (5). Membranes were examined by phase contrast microscopy for morphological alterations. Table I shows the results of these studies. Examination of several samples of blood from different species permits some general conclusions to be drawn concerning the behavior of their membranes under different isolation conditions. Human erythrocyte membranes appeared to be the most stable under all isolation conditions. Cow erythrocyte membranes tend to crenate badly in the phosphate preparation. Dog and cat membranes are particularly susceptible to fragmentation in Tris. All membranes tend to fragment or crenate during prolonged or repeated washing with hypotonic buffers. Extensive washing to remove traces of hemoglobin is therefore undesirable if one wishes to retain an intact membrane. Polyacrylamide gel electrophoresis. Although the application of gel electrophoresis in SDS to erythrocyte membrane fractionation has been described in considerable detail in earlier publications (3,18, 19), several features that have been noted in this work are worthy of mention. The SDS electrophoretic method is quite versatile in its application and can be used under a variety of conditions. In our experiments the SDS concentration in the gel and in the electrophoresis

477

PROTEINS TABLE

VARIATION

Species

I

OF MEMBRANE MORPHOLOGY PREPaRATION TECHNIQUE Preparation method

Human Dog

Phosphate Phosphate

Cat cow

Phosphate Phosphate

Pig

Phosphate

Sheep Human

Phosphate Tris

Dog

Tris

Cat cow

Tris Tris

Pig Human

Tris Ca2+-Verona1

Dog Cat cow

Cae+-Verona1 Ca2+-Verona1 Ca2+-Verona1

Pig Sheep

Ca2+-Verona1 Ca2+-Verona1

WITH

Morphology

Mostly biconcave Some biconcave, some round Round Round, badly crenated Round, badly crenated or fragmented Round Mostly round, some stromalytic forms Round, many fragments Very fragmented Round with some spikes Very fragmented Mostly round, some biconcave Round Round Round, somewhat crenated Round Round and crenated

buffers have been varied from 0.1% to 1%. The electrophoretic patterns are essentially the same under these conditions if sufficient SDS is used to solubilize the samples. Samples dissolved in 24% SDS can be placed directly on gels containing either 0.1% or 1% SDS without significant aberration of the patterns. Two advantages accrue from the higher concentrations of SDS used in sample preparation. Solubilization and disaggregation are more nearly complete, and protease activity is inhibited (19). Dialysis of the solubilized samples to remove excess SDS is therefore unnecessary and undesirable, since it may result in reaggregation or proteolysis. Heating the samples to disaggregate (18) is not necessary, but does aid in destroying any protease activity present in the sample. We routinely reduce samples with fresh mercaptoethanol (1%) after heating to prevent disulfide formation, even if the samples have been previously reduced. The use of 0.1%

478

ET AL.

KOBYLKA

SDS in gels has been preferred because of the enhanced resolving power and the facilitation of the staining and destaining. Destaining in the presence of methanol speeds the removal of SDS from the gel. Failure to remove the SDS retards the uptake of stain by proteins even if they are fixed within the gel. Electrophoretic analyses have also been performed using the complete protocol of Lenard (18) and the staining technique of Fairbanks et al. (19). No significant variations from our own results were noted in the number or distribution of bands except in was cases where enhanced resolution achieved as noted in the results to follow. Protein com,parisons. The SDS electrophoretic procedure has been used to analyze the proteins from erythrocyte membranes prepared by three different methods. Figure 1 shows t’he patterns obtained by scanning the gels from membranes prepared by the Ca2+Verona1 procedure of Burger et al. (5). Contrary to our results reported earlier with a similar group of species prepared by a different procedure (a), the major bands for each species are almost identical to those for the other species. Species specific bands do ap-

CAT

A

pear for the sheep (mol wt 126,000), dog (45,000) and pig (57,000), as reported earlier by Lenard (3). The bands in the gels have been designated as nine separate band areas (I-IX), which can be identified for each species. The band at the far right on each gel scan results from residual hemoglobin in the membrane. Comparable gel scans are shown in Figs. 2 and 3 for the membranes prepared by the phosphate and Tris procedures, respectively. Although the bands have not been as clearly resolved due to a shorter electrophoresis time, the band areas can be assigned as in Figure 1 by comparing molecular weights of the bands in the three different scans. Some differences can be noted in the protein patterns for membranes prepared by different methods. For example, the specific band (mol wt 57,000) of pig membranes shows a greater dominance over the other bands in the phosphate and Tris preparations. Demus and Mehl (20) have previously noted a predominant band in pig membranes which probably corresponds to this. Some other examples of variations in relative band intensities can be noted for the different preparations, but the most striking observa-

I

PIG

DOG

MIGRATION FIG. 1. Gel scans of coomassie blue branes prepared by the veronal-Cae+ was applied to each 6% acrylamide gel Gels were stained and destained in 7%

DISTANCE

(cm)

stained polyacrylamide gels of erythrocyte memprocedure (5). Approximately 100 pg of protein in 3% SDS (solubilieed from lyophilized materials). acetic acid as described.

ERYTHROCYTE

MEMBRANE

479

PROTEINS

tion occurs with dog membranes prepared in Tris-EDTA. In this case the protein profile has been completely altered. In earlier experiments in which membranes were prepared

r

PIG

CAT i

CAT DOG

DOG HUMAN

HUMAN

8 4 6 2 MIGRATION DISTANCE km)

IO

FIG. 3. Gel scans for membranes prepared by Tris-EDTA procedure. Electrophoresis conditions equivalent to those of Fig. 2. 4 6 8 MIGRATION DISTANCE (cm)

IO

FIG. 2. Gel scans of coomassie blue stained polyacrylamide gels of erythrocyte membranes prepared by the phosphate procedure (4). Electrophoresis conditions are the same as in Fig. 1 except for a shortened time of electrophoresis.

by the phosphate procedure and washed extensively to remove residual traces of hemoglobin, both dog and cat membranes showed significant variations from the others (2). Amino acid compositions of membranes prepared in phosphate and Tris were shown to

480

KOBYLKA

El’

AL.

be quite similar. Variations between species Fig. 4. Equivalent amounts of membranes are no more pronounced than variations be- were then dissolved in 2 (;a SDS and subtween t’he two types of preparat’ions, even jected to electrophoresis. The gels were for Tris-EDTA dog ghosts, which show a scanned to yield the results shown in Fig. grossly different protein profile. Two possibilities suggest themselves to cxI I I plain the gross alteration of Tris-EDTA dog A membrane protein patterns, the loss of membrane proteins during hemolysis and washing and the degradation of membrane proteins by proteolysis. Several observations indicate that the latter explanation is more likely correct. (1) The altered profile shows the loss of higher molecular weight components and the appearance of new bands at intermediate and lower molecular weights; these observations would not be possible without a decrease in the yield of membrane materials, which was not observed. (2) The changes in the protein pattern are similar to those observed for intact membranes treated with low concentrations of proteolytic enzymes (21). (3) Membrane samples which are dissolved by heating in SDS still show significant amounts of the band I (spectrin) of the unmodified preparation, but samples which are dissolved at room temperature in SDS show an absence of this band (as in Fig. 3). This suggests the presence of a protease which is still active in the solubilizing medium. (4) No significant C changes are noted in the amino acid composition of the altered membranes when compared to membranes of other species or membranes of the same species which have normal electrophoretic patterns. Complete removal of the larger polypeptide chains would be more likely to alter composition than proteolysis. Proteolytic enzyme digestion t’hat results in the cleavage of every cchain in the membrane causes little loss of l 2 3 4 membrane peptides5, most of which are apMIGRATION DISTANCE (cm) parently tight,ly bound into the membrane FIG. 4. Effects of removal of white blood cells structure. on erythrocyte membrane proteins. A, membranes The source of the proteolytic activity prepared from cells which were not freed of white should be considered if it is to be eliminated cells by removal of buffy coat. B, membranes preas a problem in membrane preparations. pared from cells washed to remove buffy coat. No Fairbanks et al. (19), have suggested that precautions taken to remove white cells that reproteases may be derived from contaminatmained with membrane preparation. C, membranes prepared from cells washed to remove huffy ing white blood cells. To test this hypothesis human erythrocyte ghosts were prepared un- coat. Tight cell pellet removed from each memder three sets of conditions, described in brane washing step to eliminate residual white cell 6R. B. Triplett,

unpublished

observation.

contamination (19). Electrophoresis 6 cm, 5% acrylamide gels.

performed

on

ERYTHROCYTE

MEMBRANE

4. Clear differences can be seen between the membranes which were not contaminated with white cells (Fig. 4C) and those which were (Figs. 4A and 4B), even though the samples were run on 6 cm gels and do not show highly resolved bands. The contaminated samples show a decreased amount of high molecular weight components (I-IV), a shift of the pattern toward lower molecular weights and a decreased resolution. These are phenomena similar to those observed with dog cell membranes prepared by the Tris-EDTA method, although the effects do not appear as severe since the membranes were not fragmented by the preparation technique. Further confirmation of the effects of white cell proteases on erythrocyte membranes was obtained by the incubation of small aliquots of the white cells isolated from red cell preparations with intact erythrocyte membranes. The effects were similar to those described in Fig. 4. In addition it was shown that the addition of low concentrations (0.1%) of SDS enhanced the degradation of the membrane proteins. These experiments show clearly that the white cell proteases are a source of the problem of membrane protein degradation. One of the major questions to be answered about membrane structure concerns the number of polypeptide chains present in the membrane. Although no technique can be used alone for the absolute determination of the number of chains because of possible overlaps, the electrophoresis can be manipulated to achieve maximum resolution. Two techniques have been used in studying human and bovine erythrocyte membranes, electrophoresis on long gels (12-15 cm) and elect’rophoresk on gels with varying acrylamide concentrations (4-10 %). Figure 5 shows the pattern for human erythrocyte membranes obtained on a 13 cm 5% gel. The amount of protein applied to the longer gels must be increased in order to obtain significant

bands

of the

lower

molecular

weight

components. Eleven major components can be clearly distinguished in this gel (Fig. 5, human), and can also be determined from experiments

using varied

percentages

of acryl-

amide. The band area designations are shown

0

481

PROTEINS

2

4

6

MIGRATION

DISTANCE

8

IO

(cm)

FIG. 5. Top, electrophoresis of bovine erythrocyte membranes on 13.cm 57, gels, showing resolution of component III into two bands (100 pg protein applied). Bottom, electrophoresis of human erythrocyte membranes on 13.cm 5% gels, showing nine band areas and resolution of component IV (350 pg protein applied). Running times and chart widths of two examples are not identical, resulting in slight apparent displacement of bands when comparing profiles. Both were stained by procedure 3 using 9% methanol and 0.05y0 coomassie blue and 7y0 acetic acid in staining procedure.

for these components in Table II along with their molecular weights. This number of components would represent a minimum number for the polypeptide chains, although it should be realized that all of these may not be involved in membrane structure. Band IX, for example, can be removed by further washing in hypotonic buffers, has a 25,000 mol wt and occasionally shows a slight brownish color before staining. It may be identical to the erythrocyte hemoprotein recently reported by Hultquist et al. (22), and may not be a membrane component. Both bands I and IV can be resolved into at least two components for membranes of most species, but band IV appears to be a single component in pig membranes. The molecular weights of the two components of band I, called spectrin by Marchesi and Steers (23), have been variously reported from 140,000 to 300,000. On SDS electrophoresis these components run slightly behind myosin with a molecular weight reported variously at

482

KOBYLKA

200,000-220,000 (8, 12). Band III is of considerable interest because of its asymmetric appearance caused by a diffuse trailing edge, which suggests the possibility of two or more interacting components in this band (19). By decreasing the concentration of material applied to a long gel to 100 pg, one can visually detect two bands in this area for the bovine erythrocyte membrane. These are shown clearly in the gel scan of Fig. 5. It should also be noted that the tailing phenomenon has now been eliminated. Two components in band III have also been noted in human erythrocyte membranes which have been treated with low concentrations of trypsin.5 Extraction of certain protein components can be achieved for both bovine and human erythrocyte membranes. Figure 6 shows that bands I and VII are extracted by dialysis against EDTA at pH 9.5 and 4’. In the presence of 0.3 M NaCl extraction of both of these components is suppressed, and band VIII is preferentially removed. Fairbanks et al. (19), have also recently shown that these components can be extracted speccifically, although the extraction method varied considerably from that reported here. No conclusive evidence has been presented to show that the bands of SDS electrophoresis are discrete polypeptide chains rather than aggregates of smaller chains. In fact several groups have reported disaggregation of the larger chains of the membrane polypeptides into smaller components. In each case these results appear to derive from proteolysis (24, 25) and from misinterpretation of labeling (26) or staining (27, 28) patterns or a combination of these problems. Experiments which show complete disaggregation by heating or incubation with denaturing agents before SDS electrophoresis (18, 19) are inconclusive because the reagents were removed before electrophoresis, allowing the possibility of recombination. Experiments in our laboratory have shown that electrophoresis in the presence of 8 M urea (in both sample and gel) does not indicate any disaggregation of the major polypeptide chains. Further evidence of the components’ disaggregated nature is found by comparing the results with molecular electrophoresis weights obtained by chromatography in 6 M guanidine hydrochloride (29). The distribu-

ET AL.

FIG. 6. Extraction of proteins from bovine erythrocyte membranes.Gels contain (left to right), untreated bovine erythrocyt,e membranes; supernatant of membranes extracted by 44-hr dialysis at 4’ against 0.3M NaCI, 0.5 mM EDTA, 5 mu mercaptoethanol ; precipitate of NaCI-EDTA extraction; supernatant of membranesextracted by 44-hr dialysis at, 4”against 0.5 mMEDTA and 5 mu mercaptoethanol ; precipitate of EDTA extraction. Samples were dialyzed against water and lyophiliced before electrophoresis. Approximately 100pg of protein applied to each 10 cm gel. Electrophoresis and staining those of Fig. 5.

conditions

are equivalent

to

tion of molecular weights is quite similar (Table II) when one considers the lower resolving power of the column chromatography and the fact that band III is not extracted by guanidine hydrochloride (19). Glycoproteins of thR membrane. The glycoproteins of the erythrocyte membrane can also be studied by polyacrylamide gel electrophoresis, but the conditions for detection are more critical than for the proteins. Fail-

ERYTHROCYTE TABLE

MEMBRANE

II

MOLECULAR WEIQHT OF ERYTHROCYTE MEMBRANE PROTEINS Component band

I II III IV V VI VII VIII IX

Mel wt X 10-J Guanidine SDS Electrophoresis” chr,,motographyb

225 160 112 86 69 49 40 31 26

483

PROTEINS

of acrylamide in the gels used. A similar phenomenon has also been noted in other laboratories (31, 32) using the human glycoprotein and its tryptic glycopeptide. Plots of the cal-

I

I SHEEP

I 192

74 47 29 PIG

a Membrane weights of components I-III determined on 5oj, gels, IV-IX on 6% gels as described in experimental section. b Reported by Gwynne and Tanford (29).

m-e to remove SDS from the gels before staining by the periodate-Schiff procedure can result in anomalous patterns. Figure 7 shows the carbohydrate staining profile of gels of erythrocyte membranes from 5 different species on 5 % gels which were washed overnight in 15 % acetic acid to remove SDS before staining. Each of these shows a single major glycoprotein band with no component corresponding to 108,000 mol wt (3). The diffuse trailing edge of the bands is a characteristic of this staining procedure, is not always reproduced and does not appear to represent a second component except in the case of the horse, where two closely spaced components are routinely seen. The diffuse nature of the band may represent heterogeneity of the glycoprotein molecules and their incomplete interaction with SDS. If SDS is not removed from the gels and they are fixed and stained by the procedure of Zacharias et al. (30), a rather strange pattern emerges. All bands appear to stain initially, but extensive washing removes most of the stain until band III appears most heavily stained. The molecular weight values calculated from SDS electrophoresis in 5 % gels were as follows: bovine, 285,000; human, 89,000; pig, 78,000; horse, 56,000; and sheep, 42,000. However, these do not represent the true molecular weight values for these proteins, since it was shown that the calculated molecular weights depended upon the percentage

I

HUMAN

0 MIGRATION

DISTANCE (cm)

FIG. 7. Carbohydrate staining profiles of SDS acrylamide electrophoretic gels of erythrocyte membranes of various species. Gels (10 cm) were washed 24 hr with 15% acetic acid at room temperature before staining by PAS procedure.

484

KOBYLKA

culated molecular weights against gel percentage over a range of 5-10 4’0 acrylamide show different molecular weights for each species at each percent.age, showing clearly that each species has a different glycoprotein. Variations in t,he shapes of the curves for these plots indicated that some factor other than molecular weight was influencing the glycoprotein migration. Neuraminidase treatment of the membranes before electrophoresis resulted in a decrease in the mobility of the human and bovine glycoproteins in 5 % gels. For the bovine membranes the decreased mobility was observed by both PAS staining and specific labeling of the protein with radioactive diazotized sulfanilic acid (21). These results indicate that the negative charge of the sialyl residues can influence glycoprotein electrophoretic migration as well as glycopeptide migration (31). Further analyses of the membrane proteins and glycoproteins have been performed by gel permeation column chromatography in SDS solutions. Fractionat,ion over the entire range of membrane proteins can be achieved by chromatography in 1% SDS on Sepharose 4B. The chromatographic profiles for bovine erythrocyte membranes are shown in Fig. 8. Both carbohydrate and protein have been monitored to permit comparison with the molecular weight determinations made by SDS electrophoresis. Four column fractions were obtained by combining tubes and were subjected to electrophoresis in the SDS system in order to compare peaks from

100

140 FRACTION

180 NUMBER

220

FIG. 8. Column chromatographic elution profile for protein (-----) and carbohydrate (---) of bovine erythrocyte membranes on Sepharose 4B column (2.5 X 90 cm) in 1% SDS. Fractions (1.9 ml) were combined as follows: A, tubes 122-153; B, 154r181; C, tubes 182-194; D, tubes 195-215.

ET AL.

the column with the components previously described by electrophoresis. Figure 9 shows gel scans for column fractions which were subjected to electrophoresis. Several features of these comparisons are noteworthy. The distribution of protein components by molecular weight is comparable for the two separation methods. Fraction A contains components I, II and the glycoprotein, fraction B contains III and IV, fraction C contains traces of III, IV and lower molecular weight components which are not separated by chromatography, and fraction D contains primarily lipid together with small amounts of the lower molecular weight proteins. The carbohydrate profile shows only two peaks, a high molecular weight glycoprotein in the first fraction and the glycolipid associated with the lipid of the fourth fraction. The position of the glycoprotein is not the same in the column chromatography elution pattern as in the electrophoretic pattern when compared to the other membrane proteins. The glycoprotein appears to be slightly lower in molecular weight than component I when analyzed by column chromatography, but considerably higher in molecular weight by SDS electrophoresis. Comparisons with standard proteins gave estimates of 150,000 and 380,000 (4 % gel) for the chromatographic and electrophoretic molecular weight determinations, respectively, for the bovine glycoprotein. Amino acid analyses were performed on the separated protein fractions to show that they indeed represent different components. A similar set of combined chromatography and electrophoresis experiments was performed with human erythrocyte membranes. As expected, the results were essentially identical to those of bovine membranes with the exception of the characteristics of the glycoprotein. The position of the human glycoprotein in the chromatographic profile was again shifted relative to the other protein peaks, when compared to their positions on electrophoresis. Because of this the glycoprotein and glycolipid peaks are not clearly separated on the chromatographic profile. However, it was possible to estimate the glycoprotein molecular weight at about 26,000 by chromatography. This value is clearly

ERYTHROCYTE

MEMBRANE

PROTEINS

485

different from the results obtained by SDS electrophoresis even at high acrylamide percentages in the gel.

CB-A

DISCUSSION

CB-C

PAS-A

YIO MIGRATION

DISTANCE (cm)

FIG. 9. Gel scans of SDS acrylamide electrophoresis runs of fractions from column chromatography. Fractions are those from Fig. 8. Gels were run and stained in the manner of samples in Fig. 5 (coomassie blue). SDS was removed from the gels before PAS staining by a 40y0 methanol-7% acetic acid wash. The amounts of protein found in gel CB-D and carbohydrate found in gels PAS-B and PAS-C were insignificant.

A number of reports have shown that the erythrocyte membrane contains a heterogeneous collection of polypeptide chains, which vary in molecular weight from about 25,000 to over 290,900 (2, 18, 19, 29). Three different separation methods, SDS polyacrylamide electrophoresis, chromatography in SDS solution and chromatography in 6 M guanidine show similar results for the polypeptide chain distribution. A common group of polypeptides (at least 9) with similar individual molecular weights can be demonstrated for erythrocyte membranes of a number of different animal species. A major problem which has led to the observation of variation in the distribution of polypeptides by molecular weight can be attributed to the effects of variations in the membrane preparative procedures. Different preparative methods can lead to quite striking changes in membrane morphology, but the distribution of major polypeptide chains does not seem to be very sensitive to morphological changes in the absence of other effects. The major contributor to the alteration of polypeptide distributions appears to be proteolytic degradation of the membrane polypeptides. In the presence of proteolytic enzymes the fragmented membranes are rapidly digested (19). Therefore a combination of fragmentation by preparation procedures and proteolysis results in extensive membrane protein degradation. The results presented here show the effects of such a degradation. In support of the proposition of Fairbanks et al. (19), evidence is presented to show that contaminating white cells represent an important source of these degradative enzymes. Whether erythrocyte membrane proteases also contribute remains to be demonstrated. The results do emphasize the necessity for eliminating protease activity from membrane preparations if any protein studies are to be done. The question of protease contamination must be answered for preparative procedures for all membrane types before significant protein studies can be performed.

456

KOBYLKA

The glycoproteins of the erythrocyte membranes are not SO similar for different species as the other proteins. Each species investigated appears to have a single major glycoprotein, each with somewhat different characteristics, although the possibility that there is more than one protein of a similar electrophoretic mobility for a species cannot be ruled out. Investigation of the membrane glycoproteins is plagued by numerous experimental problems. Staining artifacts can lead to misidentification on SDS electrophoresis gels. The problem of determining the molecular weights of the human membrane glycoprotein is illustrated by comparing values obtained by SDS acrylamide electrophoresis in gels with varying percentages of acrylamide and by column chromatography in SDS. These values range from 26,000 to 90,000. The problem with SDS electrophoresis derives from the failure of the glycoprotein to bind SDS in the same stoichiometric proportions relative to its molecular weight as a normal protein (31). This is not surprising when one considers that about 60% of the total weight of this glycoprotein is carbohydrsite and that the protein carries a large negative charge from the contribution of its sialic acid residues. Removal of sialic acid from either human or bovine glycoproteins results in a decreased mobility of the protein on SDS electrophoresis, suggesting that the charge of the sialic acid does play a role in the electrophoretic mobility of the glycoprotein. A similar effect of sialic acid removal was shown by Segrest et al. (31), for the tryptic glycopeptide of the human erythrocyte membrane, although no change was seen in mobility on removal of sialic acid from the human membrane glycoprotein. The variations not’ed in the effects of sialic acid removal between different laboratories (19,31) may result from the effect of differences in electrophoresis conditions on the binding of SDS by the glycoprotein. The values reported for the subunit molecular weight of the human erythrocyte glycoprotein vary from 26,000 to 100,000 (10, 18, 32-34). The values obtained solely by electrophoretic analysis are questionable. It is interesting to note that values obtained by column chromatography in pyridine (28,000, Ref. 10) and in SDS (26,000) are similar and

ET AL.

are not greatly different from those reported earlier by ultracentrifugation (33, 34). However, the effect of the carbohydrate portion of the molecule on these determinations is not clear, so the validity of the absolute values is still in doubt. Recently Jackson et al. (36), have reported a mol wt of 55,000 based on a combination of electrophoretic studies and investigation of peptides obtained from purified human erythrocyte glycoprotein. This type of approach appears essential both to elucidate the size and nature of this protein and to illuminate some of the problems involved in the investigation of glycoproteins in general. One other protein of the erythrocyte membrane deserves special mention because of some of its unique properties described in this report. In the course of the present work component III has been shown to have the following properties : (1) stains anomalously with Schiff reagent if SDS is not removed, (2) shows a trailing band with coomassie blue that appears to result from the interaction of two related polypeptide chains of similar molecular weights, and (3) aggregates into a form that will not penetrate electrophoresis gels in the presence of high concentrations of salts or organic solvents that are used for lipid extractions, Fairbanks et al. (19), have commented on the fact that this protein is not extracted by the guanidine hydrochloride procedure of Gwynne and Tanford (29), but remains associated with the lipid material. This suggests that the protein is hydrophobic in nature. Amino acid analysis of CO~UIIHI chromatographic fraction B, which contains predominantly component III plus IV, indicates that the greatest ratio of hydrophobic to hydrophilic residues is in this fraction. Chemical labeling and enzymatic degradation studies from our laboratory (21) and others (25, 37-39) indicate that component III is partially accessible to the surface of the red cell membrane but not as readily accessible as the glycoprotein (21). Bretscher (40) and Steck et al. (38), have recently presented evidence suggesting that this component extends completely through the membrane. However, both of these studies assume that no changes in membrane organization occur in preparing ghosts from intact cells. This assumption may not be completely justified

ERYTHROCYTE

MEMBRANE

(41,42), casting some doubts on the applicability of the evidence to this problem. Regardless, the argument that component III should extend through the membrane is rather compelling because of its size, which would require that it occupy a significant volume of internal membrane space otherwise. This protein might represent a portion of the particles observed by freeze etch electron microscopy in red cell membranes. Further studies on this protein and others should be informative concerning the relation of the structural attributes of membrane proteins to their membrane locales. ACKNOWLEDGEMENTS We gratefully acknowledge the technica! assistance of Mrs. Joan Summers. This work was supported by grant GM 16,870 from the National lnstitutes of Health, American Cancer Society Grant P-563 and Institutional Grant IN-QlB and the Oklahoma Agricultural Experiment Station. REFERENCES 1. ZWAAL, R. F. A., AND VAN DEENEN, L. L. M., Biochim. Biophys. Acta 160, 323 (1968). 2. CARRAWAY, K. L., AND KOBYLKA, D., B&him. Biophys. Acta 219, 238 (1970). 3. LENARD, J., Biochemistry 9,5037 (1970). 4. DODGE, J. T., MITCHELL, C., AND HANAHAN, D. J., Arch. Biochem. Biophys. 100, 119 (1963). 5. BURGER, S. I’., FUJII, T., AND HANAHAN, D. J., Biochemistry 7, 3682 (1968). 6. MARCHESI, V. T., AND PALADE, G. E., J. Cell. Biol. 36,385 (1967). 7. SHAPIRO, A. L., VINUEL.~, E., AND MAIZEL, J. V., JR., Biochem. Biophys. Res. Commun. 28, 815 (1!)67). 8. WEBER, K., AND OSBORN, M., J. Biol. Chem. 244, 4406 (1969). 9. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 10. BLUMENFEL~D, 0. O., GALLOP, P.M., HOWE, C., AND LEE, L. T., Biochim. Biophys. Acta 211, 109 (1970). 11. MCGUCKIN, W. F., AND MCKENZIE, B. F., Clin. Chem. 4, 476 (1958). 12. KLOTZ, I. M., AND DARNALL, D. W., Science 188, 126 (1969). 13. SCHACHMAN, H. K., Cold Spring Harbor Symp., &ant. Biot. 28, 409 ((1963). 14. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F., Anal. Chem. 28, 350 (11956).

PROTEINS

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15. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1958). 16. EYLAR, E. H., MADOFF, M. A., BRODY, 0. V., AND ONCLEY, J. L., J. Biol. Chem. 237, 1992 (1962). 17. WARREN, L., J. Biol. Chem. 234, 1971 (1959). 9, 1129 (1970). 18. LENARD, J., Biochemistry 19. FAIRBANKS, G., STECK, T. L., AND WALLACH, D. F. H., Biochemistry 10, 2606 (1971). 20. DEMUS, H., AND MEHL, E., Biochim. Biophys. Acta 203, 291 (1970). 21. CARRAWAY, K. L., KOBYLKA, D., AND TRIPLETT, R. B., Biochim. Biophys. Acta 241, 934 (1971). 22. HULTQUIST, D. E., REED, D. W., PASSON, P. G., AND ANDREWS, W. E., Biochim. Biophys. Acta 229, 33 (1971). 23. MARCHESI, V. T., AND STEERS, E., JR., Science 169, 203 (1968). Biophys. Acta 183, 65 24. BERG, H. C., B&him. (1969). 25. BENDER, W. W., GARAN, H., AND BERG, H. C., J. Mol. Biol. 68, 783 (1971). 26. KIEHN, E. D., AND HOLLAND, J. J., Biochemistry 9, 1729 (1970). 27. L~ICO, M. T., RUOSLAHTI, E. I., PAPERMAST~R D. S., AND DREYER, W. J., Proc. Nat. Acad. Sci. U. S. 87, 120 (1970). 28. CARRAWAY, K. L., LAM, A., KOBYLKA, D., AND HUGGINS, J. W., Anal. Biochem., in press. 29. GWYNNE, J. T., AND TANFORD, C., J. Biol. Chem. 246, 3269 (1970). 30. ZACHARIUS, R. M., ZELL, T. E., MORRISON, J. H., AND WOODLOCK, J. J., Anal. Biochem. 30, 148 (1969). 31. SEGRBST, J. P., JACKSON, R. L., ANDREWS, E. P., AND MARCHESI, V. T., Biochem. Biophys. Res. Commun. 44,390 (1971). 32. BRETSCHER, M. S., Nature 231, 229 (1971). 33. MORAWIECKI, A., Biochim. Biophys. Acta 83, 339 (1964). 34. KATHAN, R. H., WINZLER, It. J., AND JOHNSON, C. A., J. Exp. Med. 113,37 (1961). 35. MARCHESI, V. T., Fed. Proc. 29,600 (1970). 36. JACKSON, R. L., SEGREST, J. P., AND M.\RCHESI, V. T., Fed. Proc. 30, 1280 (1971). 37. BRETSCHER, M. S., J. Mol. BioZ. 68,775 (1971). 38. STECK, T. L., FAIRBANKS, G., AND WALLACH, D. F. H., Biochemistry lo,2617 (1971). 39. PHILLIPS, D. R., AND MORRISON, M., Fed. Proc. 30, 1065 (1971). 4.0. BRETSCHER, M. S., J. Mol. Biol. 69,351 (1971). 41. ZWAAL, R. F. A., ROELOFSEN, B., COMFURIOUS, P., AND VAN DEENEN, L. L. M., Biochim. Biophys. Acta 233, 474 (1971). 42. CARRAWAY, K. L., KOBYLKA, D., SUMMERS, J., AND CARRAW~Y, C. A., Chem. Phys. Lipids, in press.