Surface peptides on intact Ehrlich ascites tumor cells

Printed in SW&~ Copyright 0 1974 by Acudmic Press, Inc. All rights of reproduction in any form rcswcrd

Experimental Cell Research 83 (1974) 344-350

SURFACE PEPTIDES ON 1NTACT EHRLICH Identification

By a Method Not Requiring R. E. GATES, M. McCLAlN

Laboratories

ASCITES TUMOR

Subcellular

CELLS

Fractionation

and M. MORRISON

of Biochemistry, St Jude Children’s Research Hospital, Memphis, Tenn. 38101, USA

SUMMARY Lactoperoxidase catalysed iodination of tyrosyl residues was used to label the exposed plasma membrane proteins in intact Ehrlich ascites tumor cells. Autoradiography of 1Z51-labeledintact cells revealed that the label was predominantly associated with the plasma membrane. When whole cells were solubilized and subjected to gel electrophoresis, two major labeled peptide classes of 100 000 and 80 000 D along with 4 minor labeled classes were found. An identical labeling pattern was obtained when plasma membranes isolated from labeled cells were solubilized and subjected to gel electrophoresis. These results demonstrate that the number of exposed plasma membrane peptides and their molecular weights can be determined without first isolating the membrane by subcellular fractionation procedures, a standard approach in most studies.

Until recently, proteins of the plasma membrane could not be studied without first isolating the membrane itself. This requires subcellular fractionation procedures which can lead to alteration or selective loss of membrane components. Moreover, methods required for isolation of the membrane are time-consuming and do not usually give high yields. Thus, relatively large numbers of cells have been required for studies of plasma membrane proteins. The method reported here can be used to determine the exposed proteins in the plasma membrane of a nucleated mammalian cell type, Ehrlich ascites tumor (EAT) cells, without prior fractionation of the cell. Two advantages of the procedure are its applicability to very small quantities of cells and its elimination of a possible loss or alteration of membrane polypeptides. Exptl Cell Res 83 (1974)

This method is based on the use of the lactoperoxidase macromolecular probe system, in which exposed proteins of the plasma membrane are labeled with radioactive iodide in a reaction catalyzed by lactoperoxidase [l, 21. The labeled proteins can then be fractionated by gel electrophoresis without first isolating the plasma membrane.

MATERIALS

AND METHODS

All chemical reagents employed in these studies were reagent grade and all aqueous solutions were prepared with glass-distilled water. Dulbecco’s [3] phosphate-buffered saline (PBS) was adjusted to pH 7.40 at 4°C. Nigrosin (water-soluble) from Eastman Organic Chemicals was used to prepare a 0.2 % solution in PBS. After overnight stirring the solution was filtered and stored at 4°C. Lactoperoxidase was isolated according to Morrison & Hultquist 141and lZ51as carrier-freeyodide was purchased frbm SchwarzMann. Electrophoretically purified bovine pancreas deoxyribonuclease I, crystallized and lyophilized bovine serum albumin, and bovine erythrocyte carbonic

Surface peptides of EA T cells anhydrase were obtained from Sigma Chemical Comoanv. as was sodium dodecvl sulfate (SDS). The SDS ‘was recrystallized from ethanol, and acrylamide from Eastman Organic Chemicals was recrystallized from chloroform. Pronase, B grade, was purchased from CalBiochem, rabbit muscle phosphorylase A from Worthington Biochemical Corporation and Dade moni-trol I from Scientific Products. Biological grade glutaraldehyde (50 Y/,) was obtained from Vaughn, Inc. Tween-20 was purchased from Nutritional Biochemicals, Corp. Dextran T 500 was obtained from Pharmacia Fine Chemicals, while polyethylene glycol (mol. wt, 6 000-7 500) was obtained from Matheson. Coleman & Bell.

345

5 min. The cells were washed 3 times with isotonic saline by resuspending the cells and centrifuging as above. A portion of the washed cell pellet was diluted I : IO with water and stored at 10°C. The remainder of the pellet was used for the isolation of plasma membrane. The zinc ion method of Warren et al. [I l] was used to remove the plasma membranes from the cell. The two-phase system of Brunette & Till [I21 was used to isolate the plasma membranes; membranes suspended in the two phases were centrifuged at 3 000 g for 10 min 3 times. The isolated membranes were washed twice with 0.01 M PO, buffer at pH 7.4 by centrifuging at I 000 g for IO min. The membrane pellet was diluted I :5 with water and stored at -10°C.

Gel electrophoresis Protein concentrations were determined by the fluorescence spectrophotometric method of Fairbanks et al. [5], except that the solutions were heated to 60°C for 5 min prior to the fluorescence analysis. Standard curves for protein were run with each determination using moni-trol I as the standard. Polypeptides were separated by electrophoresis in SDS on IO “b acrylamide gels [6]. To an aliquot of the cell suspension in water or of the isolated plasma membrane in water containing 300 pg of protein was added an eaual aliauot of a solution containing 2 % SDS, 2% j-mercaptoethanol, 2 mM EDTA,-8 M urea and 0.02 M PO1 buffer (Na salts) at pH 7.2. After heating to 100°C for I min the sample was lavered on the top of gels. Care was taken to avoid bubbles. Gels were stained for protein by the method of Weber & Osborn [7], except that the Coomassie brilliant blue concentration was reduced to 0.025~~ and destaining was by washing in a solution of 5 Ib methanol and 7 % acetic acid. The distribution of radioactive label was determined by lateral slicing (2 mm slices) of stained or frozen unstained gels. The slices were counted in a gamma spectrometer. Molecular weights of the labeled polypeptides were estimated by comparing the mobility on gels stained for protein of labeled components with that of marker proteins incorporated into the sample. The marker proteins were phosphorylase A, 94 000 D; albumin, 68 000 D; and carbonic anhydrase, 29 000 D [8]. Cell preparation and membrane isolation Ehrlich ascites tumor cells were generously provided by Dr Dipak Haldar [9]. Propagation and experimental cell stocks were prepared as described by Martin et al. [IO] and contained about I.5 x IO” cells/ml. BALB/c female mice weighing 20-22 g were inoculated intraperitoneally with 0.2 ml of experimental stock. Ascitic fluid and cells were harvested 8 days after inoculation. The cells were washed in PBS by centrifuging at 60 g for 10 min and the supernatant and red blood cells were removed by aspiration. After three washes in PBS the cells were counted in the presence of 0.2 Y, nigrosin [IO]. Cells which stained were considered non-viable. After labeling, the cell suspension was diluted with an equal volume of PBS and centrifuged at 120 g for

Pronase treatment In some cases intact cells were subjected to proteolvsis after thev had been labeled. The cell susuension was diluted with an equal volume of PBS after labeling and centrifuged at 120 g for 5 min. The cells were washed 3 times with PBS by resuspending the cells and centrifuging as above. The cells were diluted to IO* cells/ml with PBS and the suspension made 50 /Lg/ml in deoxyribonuclease. This enzyme prevented the massive aggregation of cells which otherwise occurred during incubation with pronase. The cell suspension was divided equally and one part was made 50 /Ag/ml in pronase. After incubation at 37°C for 30 min equal volumes of cold PBS were added and the cells were centrifuged at 120 g for 5 min. After 3 washes with isotonic saline, portions of the cell pellets were diluted I : IO with water and stored at ~ 10°C. The remainder of the cell pellets were used to prepare plasma membrane.

The labeled cells were washed as above and then suspended in PBS containing 5 9!, glutaraldehyde at 4C for 24 h. The cells were then centrifuged and resuspended twice with glutaraldehyde-containing buffer. The cells were post-fixed with osmium tetraoxide, dehydrated with ethanol and propylene oxide, and embedded in Epon 812. Sections of 0.1 Irrn were placed on Formvar-coated grids and Ilford L-4 nuclear emulsion applied [13]. After 2 weeks the emulsion was developed with Kodak microdo]->< developer. The sections were stained with Reynolds’ [I41 lead citrate and coated with carbon film. Observations were made on a Siemens IA electron microscope.

RESULTS Localization of label on the plasma membrane

Labeled whole cells were examined autoradiographically to determine the distribution of label in the cell. A representative autoradiograph is shown in fig. 1. In 11 pictures of Exptl Cell Res 83 (1974)

346

R. E. Gates

Fig. 1. Radioautograph of lactoperoxidase catalysed lz51labeling of an intact EAT cell. Black grains indicate position of Y in the thin section through the cell. -4 ,um. Labeling was done at 1°C in PBS containing per ml: 10’ cells, IO-” moles of KI, 0.1 mCi of lz51as iodide and 10-r” moles of lactoperoxidase. Hydrogen peroxide at 0.01 M in PBS was added 10 times at 1 min intervals using 0.001 ml per ml of cell suspension for each addition.

intact cells at this magnification, a total of 71 grains were counted, and 58 were associated with the plasma membrane. Intracellular grains may not indicate labeling at their location in the cell. Following 24 h fixation with glutaraldehyde, the supernatant had 50% of the radioactivity associated with the cell pellet even though the cells had been washed 3 times prior to fixation. Thus, it appeared that all the unbound iodide could not readily be washed from the cells. When a damaged cell without an intact plasma membrane was observed on an autoradiograph the grains were randomly distributed through the damaged cell. Cell debris in the preparation was characterized by a dense distribution of grains. Because of these Exptl Cell Res 83 (1974)

observations, only cell preparations with at least 96 % viability determined by dye exclusion immediately prior to iodination were used. Determination of labeled peptide molecular weights Solubilization of the labeled whole cells or of membranes isolated from these cells in SDS, followed by electrophoresis on 10% acrylamide-SDS gels separated the polypeptides from the cell or the membrane on the basis of size. The upper plots in fig. 2 show the distribution on gels of the labeled polypeptides in the whole cell and the membrane, respectively. With both preparations the molecular weights of the two major labeled

Surface peptides qf EAT cells

A

341

B

Fig. 2. Abscissa: mol. wt ( x IO-“); ordinate: cpm/slice ( x lo-%). Gel labeling pattern of rzsI labeled intact EAT cells which were incubated after labeling without pronase (untreated) or were incubated with pronase. Gel origins on the left. Background levels have been subtracted. (A) Whole cell electrophoresis; (B) isolated plasma membrane electrophoresis. Labeling was done at 1’C in PBS containing per ml: IO8cells, 0.4 mCi 9 as iodide and 10-r” moles of lactoperoxidase. Hydrogen peroxide at 0.01 M in PBS was added 5 times at 2 min intervals using 0.002 ml per ml of cell suspension for each addition.

peptide classes were 100 000 and 80 000 D. There were 4 minor labeled peptide classes with respective mol. wts of 60 000, 55 000, 45 000 and 40 000 D. The pronounced labeling at the top of the gel from whole cell electrophoresis (fig. 2A) is not considered a separate labeled peptide class because its magnitude varied markedly depending on the sample and gel preparation. In some cases the amount of label at the top of the gel was less than 2/3 of the label in the 100 000 D range [15]. Similar variability was observed with the first peak on gels from membrane electrophoresis (fig. 2 B). These variable results obtained at or very near the gel origin suggested an artifact due to irreversible aggregation of exposed membrane proteins [5]. The upper labeling patterns in fig. 2 represent the untreated controls for pronasetreated cells. Identical patterns were obtained when the incubation with deoxyribonuclease was omitted.

Characterization of plasma membrane preparation

Comparison of the upper labeling patterns in fig. 2 provided a unique means of characterizing the plasma membrane preparation. Labeled cells and plasma membranes isolated from these cells were each electrophoresed using equal amounts of proteins. The total cpm in the membrane gel divided by the total cpm in the whole cell gel gave the increase in spec. act. for labeled peptides in the membrane preparation. Assuming that only the exposed proteins of the plasma membrane are being labeled, this increase in spec. act. is a direct measure of the degree of purification of the plasma membrane. By this calculation we obtained a six-fold degree of purification. The identical appearance of the two labeling patterns provided strong evidence that membrane components had not been altered or lost selectively as a result of membrane isolation. Exptl Cell Res 83 (1974)

348 R. E. Gates Determination of pronase treatment yffect Intact cells were treated with pronase after labeling. This procedure did not destroy the cells, since, as judged by dye exclusion, 94 0/h of the cells were viable after exposure to the enzyme. As shown in fig. 2, 65 ?,) of the label was removed by pronase treatment. Because the label was not removed selectively, we concluded that all labeled peptide classes were equally susceptible to the proteolytic enzyme. The percent of label removed over a IO-fold concentration range in pronase remained constant at 65 9,. To establish that the observed labeling pattern arose from exposed proteins on the outside of intact cells and not from exposed proteins on debris, the following rationale was used. The proteolytic enzyme pronase can digest those proteins on the exposed surface of the membrane and those proteins which are exposed in the broken cells. Since 95 ?, of the cells are intact, the proteolytic enzyme could have access to maximally about 5 %, of these latter proteins. Thus, 95 “/; of the intracellular protein would be inaccessible and should be very minimally altered in concentration by the pronase treatment. The pattern on the protein stained gel attributable to intracellular proteins would therefore not be changed. Fig. 3 shows the Coomassie-stained gels of membranes isolated from untreated cells and from pronase-treated cells. A stained peptide band at 100 000 D has disappeared as a result of pronase treatment. This peptide band must represent a peptide exposed on the surface of all the whole cells. Since this band coincides with the first major labeled peptide class in fig. 2, we conclude that the observed labeling pattern reflects iodination of the exposed surface proteins of the intact cell. While all labeled peptide classes were Expil

Cell Res 83 (1974)

A.

B

c

mol. wt ( 10--3). Coomassie brilliant blue stained 10 ‘!b acrylamideSDS gels from the electrophoresis of: (A) membranes isolated from untreated cells; (B) membranes isolated from pronase-treated cells; (C) untreated whole cells. Fig. 3. Ordinate:

uniformly reduced by pronase, only the 100 000 D class was seen as altered on Coomassie-stained gels. This result was not unexpected since most of the peptides seen en the gels from membrane electrophoresis were not membrane proteins. Support for this conclusion comes from the observation that pure plasma membrane proteins comprise a few percent of the total protein of the cell [16], whereas our membrane fraction represented 16 % of the cell protein, suggesting considerable contamination by nonplasma membrane protein. Indeed, comparison of the Coomassie-stained gels from

Surface peptides of EAT cells whole cells and from isolated membrane in fig. 3 showed only minor quantitative changes. DISCUSSION catalysed iodination of Lactoperoxidase intact cells labels only plasma membrane peptides exposed on the surface of intact cells [I, 21. The unique feature of this report is that these peptides can be characterized according to mol. wt and number without any subcellular fractionation. Characterizing these peptides directly from the whole cell has several advantages over methods involving subcellular fractionation: it permits the use of very small samples and avoids possible alteration or selective loss of membrane components by either solubilization or selective isolation [16]. Use of the whole cell is feasible because the labeled polypeptides are easily determined from the incorporated radioactive iodine, which is easy to detect and quantitate. The second feature of this report is that the use of labeled cells provides a simple method of characterizing the plasma membrane preparation from a particular cell type. Comparison of the labeling patterns of whole ceils and isolated plasma membranes involves the determination of the increase in spec. act. for several different labeled peptides. If this increase is the same for all labeled components, then there is strong evidence against alteration or loss of membrane peptides in the process of isolation. This easy, simultaneous, quantitative comparison of the increase in spec. act. for several peptides presents the unique advantage of our method. Other means of characterizing plasma membrane preparations, such as quantitation of enzyme activity or of specific antigenic determinants, require that several such unambiguous markers be available and be analysed to determine if the increase in

349

spec. act. is identical for all of them. Aside from the error inherent in the determination of the increase in spec. act. for several different markers, the problem of activation or inhibition of enzyme activity or antigenic expression as a consequence of membrane isolation must be considered. Finally, the arguments used to determine specific plasma membrane markers are often circular, depending on increased levels of the marker in presumed plasma membrane preparations from several different cell types. The approach outlined in this paper does not suffer from these difficulties. In a preliminary report [I 51 the feasibility of this approach was demonstrated. Subsequently, Phillips [19] used this technique with the non-nucleated platelet. Other techniques of characterizing exposed plasma membrane proteins by lactoperoxidase catalysed iodination and whole cell analysis have been developed, but they all involve some method of fractionation. Marchalonis et al. [17] have solubilized labeled cells in 9 M urea and then removed insoluble cell materials. In this approach these investigators made no effort to evaluate the possible selective loss of labeled components into the insoluble fraction. Baur et al. [18] have determined the exposure of specific cell components by labeling intact cells, followed by solubilization and precipitation with specific antibody. This approach is satisfactory when one is attempting to determine the labeling of well-defined proteins. It is, however, not feasible for determining all the labeled components. Also, the possibility of exposure and therefore labeling of the specific component only on debris must be considered with this method. As discussed in the results, we have direct evidence that the major labeled peptide classes in EAT cells are indeed exposed on the surface of intact cells and are not exposed only in debris. Exptl Cell Res 83 (1974)

350 R. E. Gates Labeling of plasma membrane proteins exposed on the surface of various cell types has shown that only a few major classes of peptides are exposed on each cell. The human red blood cell has only two exposed proteins [20, 21, 221 as determined by different surface labeling agents. Lactoperoxidase catalysed iodination of platelets has labeled 3 major and 4 minor peptide classes [ 19, 23, 241. In L cells [25], one major component very near the origin in gel electrophoresis was reported as labeled. That labeled peptide class may be similar to the variable high mol. wt component observed in this work. This work on the Ehrlich ascites tumor cell has shown labeling of 2 major and 4 minor component classes. The method outlined should be of great use to those involved in isolation and purification of plasma membrane from most cells. It provides a convenient means for the localization of the plasma membrane fraction and also enables a determination of the degree of purification. Further, with this technique, assessment of selective loss, fragmentation or modification of membrane components is readily determined. This work was supported in part by Damon Runyon grant number 1132 and USPHS grants CA-08480 and CA- 13534. A preliminary report of this work has been presented. (Fed proc 31 (1972) 412.) This paper is dedicated to the memory of Dr Leon Journey who died on December 7, 1972. Dr Journey assisted in obtaining the radioautographs in this study.

Exptl Cell Res 83 (1974)

REFERENCES I. Phillips, D R & Morrison, M, Biochem biophys

res commun 40 (1970) 284. 2. - Biochemistry 10 (1971) 1766. 3. Dulbecco, R & Vog t,M, J exptl med 99 (1954) 167. 4. Morrison, M & Hultquist, D E, J biol them 238 (I 963) 2847. 5. Fairbanks, G, Steck, T L & Wallach, D F H, Biochemistry 10 (1971) 2606. 6. Lenard, J, Biochemistry 9 (1970) 1129. 7. Weber, K & Osborn, M, J biol them 244 (1969) 4406. 8. Neville, D M, J biol them 246 (1972) 6328. 9. Haldar, D & Freeman, K B, Can j biochem 46 (1968) 1009. 10. Martin, E M, Malec, J, Sved, S & Work, T S, Biochem j 80 (1961) 585. II. Warren, L, Glick, M & Nass, M K, J cell physiol 68 (1966) 269. 12. Brunette. D M & Till. J E, J membrane biol 5 (1971) 2i2. 13. Caro, L G & van Tubergen, R P, J cell biol 15 (1962) 173. 14. Revnolds. E S. J cell biol 17 (1963) 208. 15. Morrison; M 8( Gates, R E, The molecular basis of electron transport (ed J Schultz) vol. 4, p. 338. Academic Press, New York (1972). 16. Steck, T L, Membrane molecular biology (ed C F Fox & A Keith) p. 76. Sinauer Associates, Stanford, Conn. (1972). 17. Marchalonis, J J, Cone, R E & Santer, V, Biothem j 124 (1972) 921. 18. Baur, S, Vitetta, E S, Sherr, C J, Schenken, I & Uhr, J W, J immunol 106 (1971) 1133. 19. Phillips, D R, Biochemistry II (1972) 4582. 20. Phillips, D R & Morrison, M, FEBS letters 18 (1971) 95. 21. Bretscher, M S, J mol biol 58 (1972) 775. 22. Hubbard, A L & Cohn, Z A, J cell biol 55 (1972) 390. 23. Barber, A J & Jamieson, G A, Biochemistry IO (1971) 4711. 24. Nachman, R L, Hubbard, A & Ferris, B, J biol them 248 (1972) 2928. 25. Poduslo, J F, Greenberg, C S & Glick, M C, Biochemistry 1I (1972) 2616. Received June 15, 1973