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HeLa cell plasma membranes
Experimental Cell Research 93 (1975) 245-251
HeLa
CELL
PLASMA
MEMBRANES
Changes in Membrane Protein Composition during the Cell Cycle S. JOHNSEN, T. STOKKE and H. PRYDZ Institute
of Medical Biology,
Universify
of Tromw,
Tromw,
Norway
SUMMARY HeLa cells grown in suspension culture were synchronized by amethopterin block and thymidine reversal. In some cases an additional Colcemid block was used to obtain mitotic cells. From the various phases of the cell cycle, cells were harvested and the plasma membranes isolated. The membrane proteins were solubilized in sodium dodecyl sulphate and separated by gel electrophoresis in the presence of sodium dodecyl sarcosinate. About 35 protein bands, five of which were stained with periodic acid-S&8 reagent, appeared. Most of the bands were identical in all membrane preparations, but a few minor bands seemed to be associated with limited periods of the cell cycle. In particular, the cells in mitosis apparently contained plasma membrane proteins which did not occur in other phases. Amino acid analyses of the plasma membranes revealed no significant cell cycle-dependent changes in the amino acid composition.
Several attempts have been made recently during G2 and M phases by cell electroto study the role of the plasma membrane phoresis [7, 81. Studies on the chemical composition of in the regulation of normal and malignant cell growth, either by comparing trans- the plasma membrane during the cell cycle formed with untransformed cell lines or by are mainly restricted to carbohydrates, following changes which occur during the glycopeptides and glycolipids, and have cell cycle in synchronized cultures. been reported on whole cells [9], trypsiMost studies on cell cycle-dependent nates from cell surfaces [lo] and isolated changes have been concerned with the plasma membranes [ 11, 121. outer surface of the plasma membrane, the The present study deals with the protein cell surface. Receptors to plant agglutinins composition of the plasma membrane. We on normal fibroblasts are expressed espe- report some cell cycle-dependent changes cially during mitosis [ 1, 21. Increased blood in the protein composition of purified plasgroup H activity in HeLa cells during mito- ma membranes. sis [3] and decreased expression of transplantation antigen H-2 in mouse cells in MATERIALS AND METHODS S phase [4-6] have been observed. In- Culture methods creased negative net charge has been Suspensions of HeLa S3 cells were grown as dedemonstrated on HeLa and sarcoma cells scribed [13] except that 5% calf serum was used. Exptt Cell Res 9.7 (1975)
246 Johnsen, Stokke and Prydz The cell density was kept between 1x IO5 and 4~ 1P cells/ml by dilution with fresh medium every 1-2 days.
fer 0.1% SDS was replaced by 0. I % sodium dodecyl sarcosinate (Sarkosyl) (Koch-Light, Colnbrook, Bucks., UK). The concentrating gel was 20x6 mm and the separating gel 60x6 mm. Synchronization procedures Sucrose (10%) was added to increase the density of the samples. Bromphenol blue was used as running Exponentially growing cells, about 2 x lo5 cells/ml, marker. The electrophoresis was run at 20°C at I were synchronized as described by Mueller & Kajiwara 1141.The block was released bv the addition of 6 LL~ mA/gel during concentration and 2 mA/gel during separation. thymihine per lo6 cells (about 8 ;M). Seventy to 96% The molecular weights were estimated from a of the serum present during the synchronization had been dialysed against Ca*+-free Hanks’ balanced salt standard curve established with the following proteins which were solubilized and reduced under-the same solution. In some cases, Colcemid (0.2 pg/ml) was added 7 h conditions as the membrane proteins: Bovine serum albumin (Sigma, St Louis, MO), human y-globulin after thymidine release to achieve a higher percentage (Nutritional Biochemicals Corporation, Cleveland, of mitotic cells. The degree of synchrony was estimated from the Ohio), pepsin (Sigma), ovalbumin (Sigma), bovine number of cells. the concentration of DNA and the hemoglobin (Sigma), and RNase A (Sigma). Gels were fixed in 20% w/v sulphosalicylic acid and mitotic index. The cells were counted in a Coulter Counter. DNA was precipitated and washed in 0.5 M stained for proteins with Coomassie Brilliant Blue and for elvcoeroteins with the oeriodic acid-Schiff reaaent cold perchloric acid, hydrolysed in 0.5 M perchloric acid at 90°C for 20 min and determined according to (PASj according to Fairbanks et al. [19] followed by stamina with the mannesium salt of 8-anilinonanhthaBurton [IS] using 2-deoxyribose as standard. Mitotic index was determined as described by Bragger [16]. lene-sulphonic acid (Mg-ANS) to visualize the protein The cells were incubated in 0.1% sodium citrate at bands [20]. After staining, the gels were scanned at 37°C for 10 min, fixed in ethanol/acetic acid (3 : 1 560 nm in a Gilford spectrophotometer equipped with a linear transport accessory. vol/vol), and dripped onto ice-cold, wet microscopic slides. The fixed cells were stained in 2% orcein in 45 % acetic acid for 10 min and destained in ethanol. Amino acid analysis Cells were harvested for plasma membrane preparaPerformic acid oxidation was carried out according to tion at different intervals after thvmidine release. Cells Hirs [21]. About 100-600 pg protein of each membrane harvested at the moment of release are labelled “SO”, cells harvested after 4 h “S”. after 7 h “G2”. after 11 preparation was hydrolysed in redistilled 6 N HCl at h “M”, and after 15 h “G 1”. When Colcemid was 110°Cfor 24 h in closed ampoules under nitrogen. The hydrolysate was dried in vacua, washed twice with used, the cells were harvested 5 h after Colcemid addidistilled water, and dissolved in 2 ml of a 0.067 M tion (i.e. 12 h after thymidine release) and labelled citrate buffer pH 2.2 for analysis in a Jeol JLC-6AH “MC”. Amino Acid Analyzer. The tryptophan content was determined by the inIsolation of plasma membranes trinsic fluorescence of the membrane proteins. About 10 ~.a of protein was dissolved in 100 ~1 1% SDS in 0.1 Plasma membrane ghosts were isolated as described M sodium phosphate buffer pH 7.4 and then diluted to [13]. The purification was monitored by phase contrast 1 ml with the same buffer. The solution was excited at microscopy and by determination of the specific activ280 nm (bandwidth 6 nm) and emission was recorded at ity of ouabain-sensitive Na+, K+-ATPase [13]. 340 nm (bandwidth 8 nm) in a Perkin-Elmer Fluorescence Spectrophotometer MPF-3. Tryptophan was Protein solubilization used as standard. The membrane proteins were solubilized essentially according to Griffith [17]. To 100 ~1 plasma membrane suspension containing about 200 pg protein were added 10 $0.5 M dithiothreitol, 50 ~12 M Tris base, 50 RESULTS ~1 10% w/v sodium dodecyl sulphate (SDS) and 10 ~1 5% w/v disodium-EDTA. The samples were incubated at 100°C for 2 min, 200 mg urea was added, and Synchronization the samples subsequently incubated at 37°C for 3 h. amethopterin-adenosine-thymidine The reduced thiol groups were alkylated by the addi- The tion of 50 ~1 0.5 M 2-iodoacetamide in 1 M NaHCOn. treatment resulted in partially synchronous All samples were dialysed overnight against the upper cell populations through the S phase and electrophoresis buffer at room temperature.
Polyacrylamide
gel electrophoresis
Gel electrophoresis was performed in the discontinuous sulphate-borate buffer system described by Neville [18] with 11% acrylamide in the separating gel and running pH 9.5. In the upper electrophoresis bufExptl Cell Res 93 (1975)
the subsequent mitosis. After thymidine addition DNA synthesis started immediately and lasted for the next 7 h. A 75235% increase in DNA concentration was regularly observed. The increase in cell number
HeLa cell plasma membranes
247
[lo]. Such membranes appeared highly purified when scrutinized in the electron microscope [ 131. Gel electrophoresis
Fig. 2 shows the electrophoretic patterns obtained with proteins from plasma membranes isolated at different times after thymidine reversal. The electrophoretic resolution was slightly variable in repeated runs of the same material, but the number of bands and their intensity were quite reproducible. Each gel had about 35 bands, the most prominent with a mobility of 0.5 1 relative to bromphenol blue. Several minor bands are c -16 -a 0 4 a 12 16 20 reproduced as shoulders to the major bands due to lack of resolution during the spectroFip. 1. Abscissa: time (hours): ordinnre: (left) DNA photometric scanning although they were (G/ml); (right) cell no. (X lo-Gnl). ~” ’ la) Variations in cell number and amount of DNA in clearly resolved by inspection. The major a dell culture synchronized by amethopterinthymidine. Reversal with thymidine at time 0; (b) bands appeared at the same positions, irvariations in cell number and amount of DNA in a cell respective of the time of harvesting the culture synchronized by amethopterin-tbymidine, combined with Colcemid block from 7 to 12 h after cells. Hence, using these bands as markers, thymidine reversal. O-O, Cell number; A-A, DNA. it was possible by inspection to observe some rather small but reproducible dissimibegan J-S h after thymidine addition and larities in the minor bands of plasma membrane preparations from various parts of the continued 12-13 h after reversal (fig. la). Some 7040% of the cells doubled in this cell cycle. These minor band changes are period, but the mitotic index never ex- not so easily observable in the spectroceeded 20 % at any given moment. Addition photometric scans, due to lack of resoluof Colcemid increased the mitotic index to tion. Their locations are therefore given by about JO%. The effect of the combined arrows in fig. 2. The most marked specific band had a amethopterin-adenosine-thymidine-colcemid treatment was fully reversible. The relative mobility of 0.55. This band apcells completed mitosis and division when peared only in G2, M and M preparations and was totally absent in the other preparaColcemid was removed (fig. 1b). tions. Band 0.39 was small and in some runs almost hidden in the neighbouring band but Isolation of plasma membranes seemed to be specific for G2. The other The purity of the plasma membrane fraction bands that show significant cell cycle phase was evaluated as described [13]. The in- changes are not completely specific in the crease in specific activity of ouabain-sensi- sense that they are absolutely absent in certive Na+, K+-ATPase compared with the tain phases and present in others, but whole homogenate was regularly 12-20-fold marked differences in the relative inten-
248
Johnsen, Stokke and Prydz
, Fig. 2.
0.2
0.4
0.6
0.8
1 .o
0.2
0.4
0.6
sities of certain bands were observed. Band 0.36 increased markedly in M, and G 1 preparations, band 0.46 was intensified in G 2 and M preparations, and band 0.60 in M preparations. These changes have been reproduced several times by gel electrophoresis on different plasma membrane preparations. For example, S and M preparations can easily be distinguished on the basis of their electrophoretic gel patterns. The cell cycle-specific bands were observed in the central part of the gel, which gave the highest resolution. No certain changes were observed in the proximal or distal parts of the electrophoretic patterns, either because there were none or because of the poor resolution in these parts of the gel. Molecular weight estimates for the various protein bands were obtained from a standard curve established with identically treated proteins of known molecular weights. For most proteins dissolved in Sarkosyl a linear relationship was found between mobility and the logarithm of the molecular weight (fig. 3). Ribonuclease migrated further than expected from the Exptl Cell Res 93 (1975)
0.8
1.0
Abscissa:
rel.
mobility; ordinate:
Sodium dodecyl sarcosinate-polyacrylamide gel electrophoresis of plasma membrane proteins prepared from cells in various phases of the cell cycle. The gels were stained with Coomassie Brilliant Blue and scanned at 560 nm. Changing bands are indicated by arrows and relative mobilities. For explanation of SO, S, G2, M, M, and G 1, see Materials and Methods.
molecular weight. When 0.1% Sarkosyl was used the most prominent band corresponded to a molecular weight of 22000, whereas the ‘new’ band occurring in G 2, M and M, preparations corresponded to a polypeptide of 18000. PAS-staining for glycoproteins revealed 5 bands (fig. 4) which appeared in all plasma membrane preparations with no significant differences in relative intensity. In addition, strong PAS-positive material, presumably glycolipids [22] was found in the buffer front. The most prominent among the PAS-positive bands had a relative mobility corresponding to a molecular weight of about 90 000. Glycoproteins may, however, behave differently from standard proteins, in detergent gel electrophoresis. The localization of this band corresponded to a major protein band as revealed with Mg-ANS superstaining. The remaining PAS-positive bands appeared in a region of the gel too crowded with protein bands to establish whether or not the PAS-positive bands and the protein bands were identical or not.
249
HeLa cell plasma membranes
branes and the increase in glycine content from 7.6 residues/100 in S phase membranes to 9.6 in M phase preparations.
80 60 40 -
DISCUSSION 20 (7)A
0.2
0.4
0.6
0.8
1.0
3. Abscissa: rel. mobility; ordinate: mol. wt (X 10-q. Semilogarithmic plot of molecular weight against mobility relative to that of bromphenol blue for various standard proteins in sodium dodecyl sarcosinatepolyacrylamide gel electrophoresis. (I) Bovine serum albumin; (2) immunoglobulin Cl, heavy chain; (3) ovalbumin; (4) pepsin; (5) immunoglobulin G, light chain; (6) hemoglobin; (7) RNAse A.
Fig.
Amino acid composition
The amino acid composition of the isolated plasma membranes (table 1) was very similar in preparations from various phases of the cell cycle. Aspartic and glutamic acids added up to 20-23 residues per 100 residues. No attempt was made to determine how many of these residues were present in amide form before hydrolysis. Arginine, lysine and histidine added up to 13-15 residues. One sample from the G2 phase had a high lysine content, but this finding was not reproducible. The apolar amino acids (alanine, valine, leucine, isoleucine, proline, methionine and phenylalanine) were constantly 3940 residues per 100. This value may be too low, since only 24 h hydrolysis was used. Methionine was detected partly as methionine and partly as its oxidation products. The only differences in the amino acid composition that may appear significant were a decrease in proline content from 7.3 residues/100 in G2 phase membranes to 4.4-4.8 residues in M phase mem-
A linear relationship was found between the logarithm of the molecular weight and the distance migrated for several standard proteins submitted to electrophoresis in the presence of 0.1% Sarkosyl. This is in accordance with the results of Morgan [23], but not with those of Evans & Gurd [24]. Gel electrophoresis of plasma membrane proteins in Sarkosyl resolved about 35 protein bands. The differences observed in the electrophoretograms from different membrane preparations may reflect changes in the protein composition of the plasma membrane during the cell cycle. The mitochondrial contamination of our membrane preparations [13] is too small to be of any importance. Most changes were found prior to and during mitosis and disappeared in the postmitotic state, indicating their cyclic nature. This would seem to rule out the possibility that these changes are caused by direct influence of the syn-
L
I
0.2
,
0.4
1
I
0.6
I
I
0.8
I
I
1.0
rel. mobility; ordinate: AJOe. Sodium dodecyl sarcosinate-polyacrylamide gel electrophoresis of glycoproteins in the plasma membrane. After electrophoresis the gel was stained with PAS and scanned at 560 nm. Identical patterns were obtained in all membrane preparations.
Fig. 4. Abscissa:
Exptl Cell Res 93 (1975)
250
Johnsen, Stokke und Prydz
Table 1. Amino acid compositiona phases of the cell cycle
ofplasma
membrane preparations,from
cells in rvariou
Moles/l00 moles Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine (half) Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophanb
so 7.0 2.1 5.2 8.8 5.3 6.5 12.6 6.9 7.7 7.7 2.0 6.2 Present 4.8 8.7 1.9 3.5 1.2
S
G2
M
M,
GI
7.4 2.1 5.2 9.6 5.3 6.5 13.5 5.3 7.6 7.6 1.7 6.8 2.4 4.9 8.8 0.9 3.4 1.2
8.8 2.7 5.0 7.9 5.0 6.9 II.5 7.3 8.2 7.5 1.9 5.7 2.2 4.7 8.2 2.0 3.2 1.2
6.4 1.7 4.8 9.4 5.5 6.7 13.2 4.4 9.6 8.7 1.6 6.6 2.2 4.7 9.0 1.2 3.3 I.2
6.4 2.1 5.3 9.3 5.5 6.8 13.3 4.8 8.5 8.3 1.8 6.5 2.3 4.6 9.1 0.9 3.5 1.2
7.5 2.2 5.3 8.5 5.2 6.9 13.0 6.5 8.5 7.8 1.6 6.5 Present 4.8 8.4 I.0 3.3 1.2
a No correction for loss during hydrolysis. Each value mean of two determinations on separate samples. b Determined in spectrophotofluorimeter (see Materials and Methods).
chronizing drugs. Changes in the protein composition of the plasma membrane could be produced either by synthesis and insertion of new polypeptides or by proteolytic cleavage of proteins already present. Investigations are not in progress to examine these possibilities by means of isotope incorporation. The amino acid composition represents an average of at least 35 different polypeptide chains and it is therefore reasonable to find a rather normal distribution in the frequency of the amino acid residues. The variations in the proline and glycine values through the cycle may be significant. The fraction of hydrophobic amino acid residues found here was not markedly different from that in membrane proteins from other sources (e.g. [25-271). The average amino acid composition of intrinsic membrane proteins may be biased towards hydrophobic residues [28] but this is not revealed in analysis of whole membrane Exptl Cd/ Res 93 (1975)
preparations. Several reports conclude that the amino acid composition is not noticeably different from that of simple soluble proteins [29-311. Calculated according to Vanderkooi & Capaldi [28] our preparations had a polarity around 45% which is well within the range of soluble proteins. This work was supported by the Norwegian Research Council for Science and the Humanities (grant no.
c.07,,4-I),
REFERENCES I. FOX, T 0, Sheppard, J R &Burger, M M, Proc natl acad sci US 68 (1971) 244. 2. Shoman, J & Sachs, L, Proc natl acad sci US 69 (1972) 2479. 3. Kuhns, W J & Bramson, S, Nature (London) 219 (I%@ 938. 4. Cikes, M & Friberg, S, Proc natl acad sci US 68 (1971) 566. 5. Pastemak, C A, Warmsley, A M H & Thomas, D B, J cell biol50 (1971) 562. 6. Sumner, M C B, Collin, R C L S & Pasternak, C A, Tissue antigens 3 (1973) 477. 7. Brent, T P & Forrester, J A, Nature (London) 215 (1%7) 92. 8. Mayhew, E, J gen physio149 (1966) 717.
*
HeLa cell plasma membranes 9. Click, M C, Gemer, E W & Warren, L, J cell physiol77 (1971) I. 10. Glick, M C & Buck, C A, Biochemistry 12 (1973) 85. 11. Nowakowski, M, Atkinson, P H & Summers, D F, Biochim biophys acta 266 (1972) 154. 12. Graham, J M, Sumner, M C B, Curtis, D H & Pastemak, C A, Nature 246 (1973) 291. 13. Johnsen, S, Stokke, T & Prydz, H, J cell biol 63 (1974) 357. 14. Mueller, G C & Kajiwata, K, Biochim biophys acta 114 (1966) 108. 15. Burton, K, Biochem i 62 (1956) 315. 16. Bragger, A, Transiocation in human chromosomes. Thesis. Oslo University Press. Oslo (1967). 17. Griffith, I P, Biochem j 126 (1972) 553. 18. Neviile, D M jr, J biol them 246 (1971) 6328. 19. Fairbanks, G, Steck, T L & Wallach, D F H, Biochemistry 10 (1971) 2606. 20. Hartman, B K & Udenfriend, S, Anal biochem 30 (1969)391.
21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
251
Hirs, C H, J biol them 219 (1956) 611. Lenard, J, Biochemistry 9 (1970) 1129. Morgan, J, Exptl cell res 65 (1971) 7. Evans, W H & Gurd, J W. Biochem j 133 (1973) 189. Yu, B P & Masoro, E J, Biochemistry 9 (1970) 2909. Yang, S & Griddle, R S, Biochemistry 9 (1970) 3063. Guidotti, G, Ann rev biochem 41 (1972) 731. Vanderkooi, G & Capaldi, R A, Ann NY acad sci 195 (1972) 135. Engelman, D M & Morowitz, H J, Biochim biophys acta 150 (1968) 385. Rosenberg, S A & Guidotti, G, Red cell membrane (ed G A Jamieson & T J Greenwald) p. 93. Lippincott, Philadelphia, Pa (1969). Singer, S J, Ann NY acad sci 195 (1972) 16.
Received October 4, 1974 Revised version December 23. 1974
ExptICell
Res 93 (1975)
HeLa
CELL
PLASMA
MEMBRANES
Changes in Membrane Protein Composition during the Cell Cycle S. JOHNSEN, T. STOKKE and H. PRYDZ Institute
of Medical Biology,
Universify
of Tromw,
Tromw,
Norway
SUMMARY HeLa cells grown in suspension culture were synchronized by amethopterin block and thymidine reversal. In some cases an additional Colcemid block was used to obtain mitotic cells. From the various phases of the cell cycle, cells were harvested and the plasma membranes isolated. The membrane proteins were solubilized in sodium dodecyl sulphate and separated by gel electrophoresis in the presence of sodium dodecyl sarcosinate. About 35 protein bands, five of which were stained with periodic acid-S&8 reagent, appeared. Most of the bands were identical in all membrane preparations, but a few minor bands seemed to be associated with limited periods of the cell cycle. In particular, the cells in mitosis apparently contained plasma membrane proteins which did not occur in other phases. Amino acid analyses of the plasma membranes revealed no significant cell cycle-dependent changes in the amino acid composition.
Several attempts have been made recently during G2 and M phases by cell electroto study the role of the plasma membrane phoresis [7, 81. Studies on the chemical composition of in the regulation of normal and malignant cell growth, either by comparing trans- the plasma membrane during the cell cycle formed with untransformed cell lines or by are mainly restricted to carbohydrates, following changes which occur during the glycopeptides and glycolipids, and have cell cycle in synchronized cultures. been reported on whole cells [9], trypsiMost studies on cell cycle-dependent nates from cell surfaces [lo] and isolated changes have been concerned with the plasma membranes [ 11, 121. outer surface of the plasma membrane, the The present study deals with the protein cell surface. Receptors to plant agglutinins composition of the plasma membrane. We on normal fibroblasts are expressed espe- report some cell cycle-dependent changes cially during mitosis [ 1, 21. Increased blood in the protein composition of purified plasgroup H activity in HeLa cells during mito- ma membranes. sis [3] and decreased expression of transplantation antigen H-2 in mouse cells in MATERIALS AND METHODS S phase [4-6] have been observed. In- Culture methods creased negative net charge has been Suspensions of HeLa S3 cells were grown as dedemonstrated on HeLa and sarcoma cells scribed [13] except that 5% calf serum was used. Exptt Cell Res 9.7 (1975)
246 Johnsen, Stokke and Prydz The cell density was kept between 1x IO5 and 4~ 1P cells/ml by dilution with fresh medium every 1-2 days.
fer 0.1% SDS was replaced by 0. I % sodium dodecyl sarcosinate (Sarkosyl) (Koch-Light, Colnbrook, Bucks., UK). The concentrating gel was 20x6 mm and the separating gel 60x6 mm. Synchronization procedures Sucrose (10%) was added to increase the density of the samples. Bromphenol blue was used as running Exponentially growing cells, about 2 x lo5 cells/ml, marker. The electrophoresis was run at 20°C at I were synchronized as described by Mueller & Kajiwara 1141.The block was released bv the addition of 6 LL~ mA/gel during concentration and 2 mA/gel during separation. thymihine per lo6 cells (about 8 ;M). Seventy to 96% The molecular weights were estimated from a of the serum present during the synchronization had been dialysed against Ca*+-free Hanks’ balanced salt standard curve established with the following proteins which were solubilized and reduced under-the same solution. In some cases, Colcemid (0.2 pg/ml) was added 7 h conditions as the membrane proteins: Bovine serum albumin (Sigma, St Louis, MO), human y-globulin after thymidine release to achieve a higher percentage (Nutritional Biochemicals Corporation, Cleveland, of mitotic cells. The degree of synchrony was estimated from the Ohio), pepsin (Sigma), ovalbumin (Sigma), bovine number of cells. the concentration of DNA and the hemoglobin (Sigma), and RNase A (Sigma). Gels were fixed in 20% w/v sulphosalicylic acid and mitotic index. The cells were counted in a Coulter Counter. DNA was precipitated and washed in 0.5 M stained for proteins with Coomassie Brilliant Blue and for elvcoeroteins with the oeriodic acid-Schiff reaaent cold perchloric acid, hydrolysed in 0.5 M perchloric acid at 90°C for 20 min and determined according to (PASj according to Fairbanks et al. [19] followed by stamina with the mannesium salt of 8-anilinonanhthaBurton [IS] using 2-deoxyribose as standard. Mitotic index was determined as described by Bragger [16]. lene-sulphonic acid (Mg-ANS) to visualize the protein The cells were incubated in 0.1% sodium citrate at bands [20]. After staining, the gels were scanned at 37°C for 10 min, fixed in ethanol/acetic acid (3 : 1 560 nm in a Gilford spectrophotometer equipped with a linear transport accessory. vol/vol), and dripped onto ice-cold, wet microscopic slides. The fixed cells were stained in 2% orcein in 45 % acetic acid for 10 min and destained in ethanol. Amino acid analysis Cells were harvested for plasma membrane preparaPerformic acid oxidation was carried out according to tion at different intervals after thvmidine release. Cells Hirs [21]. About 100-600 pg protein of each membrane harvested at the moment of release are labelled “SO”, cells harvested after 4 h “S”. after 7 h “G2”. after 11 preparation was hydrolysed in redistilled 6 N HCl at h “M”, and after 15 h “G 1”. When Colcemid was 110°Cfor 24 h in closed ampoules under nitrogen. The hydrolysate was dried in vacua, washed twice with used, the cells were harvested 5 h after Colcemid addidistilled water, and dissolved in 2 ml of a 0.067 M tion (i.e. 12 h after thymidine release) and labelled citrate buffer pH 2.2 for analysis in a Jeol JLC-6AH “MC”. Amino Acid Analyzer. The tryptophan content was determined by the inIsolation of plasma membranes trinsic fluorescence of the membrane proteins. About 10 ~.a of protein was dissolved in 100 ~1 1% SDS in 0.1 Plasma membrane ghosts were isolated as described M sodium phosphate buffer pH 7.4 and then diluted to [13]. The purification was monitored by phase contrast 1 ml with the same buffer. The solution was excited at microscopy and by determination of the specific activ280 nm (bandwidth 6 nm) and emission was recorded at ity of ouabain-sensitive Na+, K+-ATPase [13]. 340 nm (bandwidth 8 nm) in a Perkin-Elmer Fluorescence Spectrophotometer MPF-3. Tryptophan was Protein solubilization used as standard. The membrane proteins were solubilized essentially according to Griffith [17]. To 100 ~1 plasma membrane suspension containing about 200 pg protein were added 10 $0.5 M dithiothreitol, 50 ~12 M Tris base, 50 RESULTS ~1 10% w/v sodium dodecyl sulphate (SDS) and 10 ~1 5% w/v disodium-EDTA. The samples were incubated at 100°C for 2 min, 200 mg urea was added, and Synchronization the samples subsequently incubated at 37°C for 3 h. amethopterin-adenosine-thymidine The reduced thiol groups were alkylated by the addi- The tion of 50 ~1 0.5 M 2-iodoacetamide in 1 M NaHCOn. treatment resulted in partially synchronous All samples were dialysed overnight against the upper cell populations through the S phase and electrophoresis buffer at room temperature.
Polyacrylamide
gel electrophoresis
Gel electrophoresis was performed in the discontinuous sulphate-borate buffer system described by Neville [18] with 11% acrylamide in the separating gel and running pH 9.5. In the upper electrophoresis bufExptl Cell Res 93 (1975)
the subsequent mitosis. After thymidine addition DNA synthesis started immediately and lasted for the next 7 h. A 75235% increase in DNA concentration was regularly observed. The increase in cell number
HeLa cell plasma membranes
247
[lo]. Such membranes appeared highly purified when scrutinized in the electron microscope [ 131. Gel electrophoresis
Fig. 2 shows the electrophoretic patterns obtained with proteins from plasma membranes isolated at different times after thymidine reversal. The electrophoretic resolution was slightly variable in repeated runs of the same material, but the number of bands and their intensity were quite reproducible. Each gel had about 35 bands, the most prominent with a mobility of 0.5 1 relative to bromphenol blue. Several minor bands are c -16 -a 0 4 a 12 16 20 reproduced as shoulders to the major bands due to lack of resolution during the spectroFip. 1. Abscissa: time (hours): ordinnre: (left) DNA photometric scanning although they were (G/ml); (right) cell no. (X lo-Gnl). ~” ’ la) Variations in cell number and amount of DNA in clearly resolved by inspection. The major a dell culture synchronized by amethopterinthymidine. Reversal with thymidine at time 0; (b) bands appeared at the same positions, irvariations in cell number and amount of DNA in a cell respective of the time of harvesting the culture synchronized by amethopterin-tbymidine, combined with Colcemid block from 7 to 12 h after cells. Hence, using these bands as markers, thymidine reversal. O-O, Cell number; A-A, DNA. it was possible by inspection to observe some rather small but reproducible dissimibegan J-S h after thymidine addition and larities in the minor bands of plasma membrane preparations from various parts of the continued 12-13 h after reversal (fig. la). Some 7040% of the cells doubled in this cell cycle. These minor band changes are period, but the mitotic index never ex- not so easily observable in the spectroceeded 20 % at any given moment. Addition photometric scans, due to lack of resoluof Colcemid increased the mitotic index to tion. Their locations are therefore given by about JO%. The effect of the combined arrows in fig. 2. The most marked specific band had a amethopterin-adenosine-thymidine-colcemid treatment was fully reversible. The relative mobility of 0.55. This band apcells completed mitosis and division when peared only in G2, M and M preparations and was totally absent in the other preparaColcemid was removed (fig. 1b). tions. Band 0.39 was small and in some runs almost hidden in the neighbouring band but Isolation of plasma membranes seemed to be specific for G2. The other The purity of the plasma membrane fraction bands that show significant cell cycle phase was evaluated as described [13]. The in- changes are not completely specific in the crease in specific activity of ouabain-sensi- sense that they are absolutely absent in certive Na+, K+-ATPase compared with the tain phases and present in others, but whole homogenate was regularly 12-20-fold marked differences in the relative inten-
248
Johnsen, Stokke and Prydz
, Fig. 2.
0.2
0.4
0.6
0.8
1 .o
0.2
0.4
0.6
sities of certain bands were observed. Band 0.36 increased markedly in M, and G 1 preparations, band 0.46 was intensified in G 2 and M preparations, and band 0.60 in M preparations. These changes have been reproduced several times by gel electrophoresis on different plasma membrane preparations. For example, S and M preparations can easily be distinguished on the basis of their electrophoretic gel patterns. The cell cycle-specific bands were observed in the central part of the gel, which gave the highest resolution. No certain changes were observed in the proximal or distal parts of the electrophoretic patterns, either because there were none or because of the poor resolution in these parts of the gel. Molecular weight estimates for the various protein bands were obtained from a standard curve established with identically treated proteins of known molecular weights. For most proteins dissolved in Sarkosyl a linear relationship was found between mobility and the logarithm of the molecular weight (fig. 3). Ribonuclease migrated further than expected from the Exptl Cell Res 93 (1975)
0.8
1.0
Abscissa:
rel.
mobility; ordinate:
Sodium dodecyl sarcosinate-polyacrylamide gel electrophoresis of plasma membrane proteins prepared from cells in various phases of the cell cycle. The gels were stained with Coomassie Brilliant Blue and scanned at 560 nm. Changing bands are indicated by arrows and relative mobilities. For explanation of SO, S, G2, M, M, and G 1, see Materials and Methods.
molecular weight. When 0.1% Sarkosyl was used the most prominent band corresponded to a molecular weight of 22000, whereas the ‘new’ band occurring in G 2, M and M, preparations corresponded to a polypeptide of 18000. PAS-staining for glycoproteins revealed 5 bands (fig. 4) which appeared in all plasma membrane preparations with no significant differences in relative intensity. In addition, strong PAS-positive material, presumably glycolipids [22] was found in the buffer front. The most prominent among the PAS-positive bands had a relative mobility corresponding to a molecular weight of about 90 000. Glycoproteins may, however, behave differently from standard proteins, in detergent gel electrophoresis. The localization of this band corresponded to a major protein band as revealed with Mg-ANS superstaining. The remaining PAS-positive bands appeared in a region of the gel too crowded with protein bands to establish whether or not the PAS-positive bands and the protein bands were identical or not.
249
HeLa cell plasma membranes
branes and the increase in glycine content from 7.6 residues/100 in S phase membranes to 9.6 in M phase preparations.
80 60 40 -
DISCUSSION 20 (7)A
0.2
0.4
0.6
0.8
1.0
3. Abscissa: rel. mobility; ordinate: mol. wt (X 10-q. Semilogarithmic plot of molecular weight against mobility relative to that of bromphenol blue for various standard proteins in sodium dodecyl sarcosinatepolyacrylamide gel electrophoresis. (I) Bovine serum albumin; (2) immunoglobulin Cl, heavy chain; (3) ovalbumin; (4) pepsin; (5) immunoglobulin G, light chain; (6) hemoglobin; (7) RNAse A.
Fig.
Amino acid composition
The amino acid composition of the isolated plasma membranes (table 1) was very similar in preparations from various phases of the cell cycle. Aspartic and glutamic acids added up to 20-23 residues per 100 residues. No attempt was made to determine how many of these residues were present in amide form before hydrolysis. Arginine, lysine and histidine added up to 13-15 residues. One sample from the G2 phase had a high lysine content, but this finding was not reproducible. The apolar amino acids (alanine, valine, leucine, isoleucine, proline, methionine and phenylalanine) were constantly 3940 residues per 100. This value may be too low, since only 24 h hydrolysis was used. Methionine was detected partly as methionine and partly as its oxidation products. The only differences in the amino acid composition that may appear significant were a decrease in proline content from 7.3 residues/100 in G2 phase membranes to 4.4-4.8 residues in M phase mem-
A linear relationship was found between the logarithm of the molecular weight and the distance migrated for several standard proteins submitted to electrophoresis in the presence of 0.1% Sarkosyl. This is in accordance with the results of Morgan [23], but not with those of Evans & Gurd [24]. Gel electrophoresis of plasma membrane proteins in Sarkosyl resolved about 35 protein bands. The differences observed in the electrophoretograms from different membrane preparations may reflect changes in the protein composition of the plasma membrane during the cell cycle. The mitochondrial contamination of our membrane preparations [13] is too small to be of any importance. Most changes were found prior to and during mitosis and disappeared in the postmitotic state, indicating their cyclic nature. This would seem to rule out the possibility that these changes are caused by direct influence of the syn-
L
I
0.2
,
0.4
1
I
0.6
I
I
0.8
I
I
1.0
rel. mobility; ordinate: AJOe. Sodium dodecyl sarcosinate-polyacrylamide gel electrophoresis of glycoproteins in the plasma membrane. After electrophoresis the gel was stained with PAS and scanned at 560 nm. Identical patterns were obtained in all membrane preparations.
Fig. 4. Abscissa:
Exptl Cell Res 93 (1975)
250
Johnsen, Stokke und Prydz
Table 1. Amino acid compositiona phases of the cell cycle
ofplasma
membrane preparations,from
cells in rvariou
Moles/l00 moles Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine (half) Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophanb
so 7.0 2.1 5.2 8.8 5.3 6.5 12.6 6.9 7.7 7.7 2.0 6.2 Present 4.8 8.7 1.9 3.5 1.2
S
G2
M
M,
GI
7.4 2.1 5.2 9.6 5.3 6.5 13.5 5.3 7.6 7.6 1.7 6.8 2.4 4.9 8.8 0.9 3.4 1.2
8.8 2.7 5.0 7.9 5.0 6.9 II.5 7.3 8.2 7.5 1.9 5.7 2.2 4.7 8.2 2.0 3.2 1.2
6.4 1.7 4.8 9.4 5.5 6.7 13.2 4.4 9.6 8.7 1.6 6.6 2.2 4.7 9.0 1.2 3.3 I.2
6.4 2.1 5.3 9.3 5.5 6.8 13.3 4.8 8.5 8.3 1.8 6.5 2.3 4.6 9.1 0.9 3.5 1.2
7.5 2.2 5.3 8.5 5.2 6.9 13.0 6.5 8.5 7.8 1.6 6.5 Present 4.8 8.4 I.0 3.3 1.2
a No correction for loss during hydrolysis. Each value mean of two determinations on separate samples. b Determined in spectrophotofluorimeter (see Materials and Methods).
chronizing drugs. Changes in the protein composition of the plasma membrane could be produced either by synthesis and insertion of new polypeptides or by proteolytic cleavage of proteins already present. Investigations are not in progress to examine these possibilities by means of isotope incorporation. The amino acid composition represents an average of at least 35 different polypeptide chains and it is therefore reasonable to find a rather normal distribution in the frequency of the amino acid residues. The variations in the proline and glycine values through the cycle may be significant. The fraction of hydrophobic amino acid residues found here was not markedly different from that in membrane proteins from other sources (e.g. [25-271). The average amino acid composition of intrinsic membrane proteins may be biased towards hydrophobic residues [28] but this is not revealed in analysis of whole membrane Exptl Cd/ Res 93 (1975)
preparations. Several reports conclude that the amino acid composition is not noticeably different from that of simple soluble proteins [29-311. Calculated according to Vanderkooi & Capaldi [28] our preparations had a polarity around 45% which is well within the range of soluble proteins. This work was supported by the Norwegian Research Council for Science and the Humanities (grant no.
c.07,,4-I),
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*
HeLa cell plasma membranes 9. Click, M C, Gemer, E W & Warren, L, J cell physiol77 (1971) I. 10. Glick, M C & Buck, C A, Biochemistry 12 (1973) 85. 11. Nowakowski, M, Atkinson, P H & Summers, D F, Biochim biophys acta 266 (1972) 154. 12. Graham, J M, Sumner, M C B, Curtis, D H & Pastemak, C A, Nature 246 (1973) 291. 13. Johnsen, S, Stokke, T & Prydz, H, J cell biol 63 (1974) 357. 14. Mueller, G C & Kajiwata, K, Biochim biophys acta 114 (1966) 108. 15. Burton, K, Biochem i 62 (1956) 315. 16. Bragger, A, Transiocation in human chromosomes. Thesis. Oslo University Press. Oslo (1967). 17. Griffith, I P, Biochem j 126 (1972) 553. 18. Neviile, D M jr, J biol them 246 (1971) 6328. 19. Fairbanks, G, Steck, T L & Wallach, D F H, Biochemistry 10 (1971) 2606. 20. Hartman, B K & Udenfriend, S, Anal biochem 30 (1969)391.
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Received October 4, 1974 Revised version December 23. 1974
ExptICell
Res 93 (1975)