Comp. Bhwhem Phy.giol., Vol. 62B, pp. 471 to 479.
0305-0491/79/0401-0471 $02.00/0
© Pergamon Press Ltd 1979. Printed in Great Britain
ISOLATION A N D CHARACTERIZATION OF THE ERYTHROCYTE SURFACE MEMBRANE OF THE SMOOTH DOGFISH, M U S T E L U S C A N I S L. WARREN,* C. A. BUCK,* J. L. RABINOWITZt and I. W. SHERMAN+ *The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104; "i'Veterans Administration Hospital, University and Woodland Avenues, Philadelphia, Pennsylvania 19104 and :~Department of Biology, University of California, Riverside, California 92521, U.S.A. (Received 7 August 1978) Al~tract--l. The plasma membrane of the dogfish erythrocyte is characterized. 2. Surface membranes were isolated from dogfish red cells. 3. Cells suspended in a hypotonic zinc chloride buffer (0.5 mM ZnCI2, 5 mM Tris-HCl, pH 8.0) tended to enucleate spontaneously. 4. By gentle homogenization relatively high yields of red cell "ghosts" were formed; these were purified using discontinuous gradients of sucrose and of glycerol solutions. Analyses of protein, carbohydrates and lipids were carried out. 5. There did not appear to be marked overall differences in the composition of the surface membrane of the nucleated dogfish erythrocyte compared to those of other species. INTRODUCTION There are numerous reports on the characterization of plasma membranes from the erythrocytes of vertebrates such as mammals (pig [Lenard, 1970; Hamaguchi & Cleve, 1972; Kobylka et al., 1972; Zwaal & Van Deenan, 19681, sheep [Lenard, 1970; Hamaguchi & Cleve, 1972; Zwaal & Van Deenan, 1968; Cabezas & Seco, 1975; Hudson, et al., 1975,1, horse [Hamaguchi & Cleve, 1972; Cabezas & Seco, 1975; Hudson et al., 1975,1, ox [Hamaguchi & Cleve, 1972; Zwaal & Van Deenan, 19681, rat [Lenard, 1970; Zwaal & Van Deenan, 1968,1, human [Hamaguchi & Cleve, 1972; Kobylka et al., 1972; Zwaal & Van Deenan, 1968; Hudson et al., 1975; Dodge et al., 1963; Bakerman & Wasemuller, 1967], dog [Lenard, 1970; Kobylka et al., 1972], camel [Eitan et al., 1976,1, cat [Kobylka et al., 1972], guinea pig [Hamaguchi & Cleve, 1972,1, rabbit [Hamaguchi & Cleve, 1972,1, goat [Hamaguchi & Cleve, 1972,1, cow [Kobylka et al., 1972; Cabezas & Seco, 1975; Hudson et al., 1975,1); birds (turkey [Cadwell, 1976], goose [Shelton, 1973-1, chicken [Zentgraf et al., 1971; Kleinig et al., 1971; Blanchet, 1974; Jackson, 1975], pigeon [Puchwein et al., 1974,1); and frogs (Mukherjee et al., 1976). The isolation of erythrocyte membranes from the lamprey has been reported (Asai et al., 1976). This communication presents a simple, rapid and effective method for the isolation of purified erythrocyte membranes from the elasmobranch Mustelus canis (smooth dogfish). In the technique employed, yields of pure membranes are high and organelle contamination is minimal. The membranes obtained by this method were analyzed for protein, carbohydrates and lipids. MATERIALS AND
METHODS
Preparation of dogfish red blood cell plasma membranes Smooth dogfish were obtained from the Supply Department, Marine Biological Laboratory, Woods Hole, Mass.
Blood was taken from the subcaudal vein of dogfish with a glass or plastic syringe fitted with an 18-gauge needle. The volume removed varied with the size of the animal, 100ml being the maximum taken from the largest (1 m in length) animals. Coagulation of blood was prevented by the addition of 1/10 volume of 0.1 M sodium citrate to the syringe. Twelve and a half ml of whole blood was layered over a 30-ml cushion of 30% (w/v) sucrose solution and centrifuged at 500 g for 10 min; plasma and leukocytes in the upper part of the tube were removed by aspiration. The pellet of red cells was washed twice in 0.9% (w/v) NaC1 (5min, 750g); the cells were then resuspended in an equal volume of 0.9% NaCI. To 10 ml of red cell suspension were added 50mg of solid NaHSO3, which served to inhibit proteases (Panyim & Chalkley, 1969) as well as to make the plasma membrane tougher. The contents of the tube were mixed rapidly and thoroughly. Thirty ml of "zinc buffer" were then added to 10ml of bisulfitetreated red cells in a large Dounce homogenizing tube (40ml, Kontes Glass, Co., Vineland, N.J.) (Warren & Glick, 1969). Zinc buffer contained 0.5 mM ZnCI2 and 5 mM Tris-HC1, pH 8.0. After standing at room temperature for 10 rain the homogenizing tube was transferred to an ice bucket and allowed to cool to 5°C. The cells were then broken with 20-40 strokes of an A pestle. Cell breakage and enucleation were monitored by phase contrast microscopy. Homogenization was stopped when 90% of the cells were broken. Twenty ml of the homogenate were layered over a double sucrose cushion (7.5 ml of 50~,/, (w/v) sucrose solution made in Tris-saline (0.125 M NaCI, 5mM KCI, 1.1 mM CaCI2, 25mM Tris-HCl, pH 7.6), 8ml of 25% (w/v) sucrose solution in Tris-saline) in a 40-ml centrifuge tube. The tube was centrifuged for 1 hr at 2500 rpm (1400 g) in a refrigerated International Centrifuge (Model PR-2). A slightly pink opalescent layer was seen at the interface between the 25 and 50% sucrose layers. This was collected with a 25-ml syringe fitted with a No. 18 needle that had its terminal 4 mm bent so that the bevel faced upward. The opalescent layer was diluted with an equal volume of Tris-saline buffer. A discontinuous glycerol gradient was made in a 40 ml centrifuge tube consisting of 10 ml of 40~o glycerol (v/v), 7.5 ml of 50% and 7.5 ml of 70% glycerol all made in Trissaline buffer. The tube was centrifuged in a PR-2 centrifuge for 2 hr at 3000 rpm (2000 g) at 4°C. A colorless opalescent 471
472
L. WARRENet al.
material just above the 70~o glycerol layer was collected with a syringe, diluted with 30 ml of Tris-saline buffer and centrifuged for 1 hr at 2500 rpm (1400 g) at 4°C in the PR-2 centrifuge. A pale white opalescent pellet was collected. The final yield of purified, intact "ghosts" (red cell plasma membranes) was usually about 25~0 of the original number of red cells as measured by hemocytometer counts. All procedures were carried out at 4°C unless stated otherwise. Purification procedures were monitored by phase contrast microscopy. All chemicals used were of commercial reagent grade. Neuraminidase (V. cholerae) was purchased from Calbiochem, La Jolla, California. Sialic acid was measured by the thiobarbituric acid assay (Warren, 1959) after hydrolysis with 0.1 N H2SO4 at 80~C for 1 hr. All other sugars were measured as their alditol acetates by gas-liquid chromatography (Lehnhardt & Winzler, 1968). Lipids were separated and analyzed by methods previously described in detail (Weinstein et al., 1969). DNA and RNA were analyzed by the methods of Dische (1930) and Mejbaum (1939), respectively. Amino acid analysis was carried out using a Beckmann amino acid analyzer by the method of Moore & Stein 11945)after 24hr hydrolysis at 105"C in 6 N HC1 in sealed, evacuated ampules. Analyses were carried out by Mr. R. Hyde at the Marine Biological Laboratory, Woods Hole, Massachusetts. Electron microscopy of purified ghosts Small pellets of dogfish red cell ghosts were fixed with 2~o (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, followed by osmium tetroxide in 0.1M cacodylate. Samples were dehydrated in ethanol and propylene oxide and embedded in Epon. Sections were cut on an MT-2 microtome (Sorvall-Dupont) and stained with a saturated solution of uranyl acetate in 50,50 ethanol and with lead citrate (Reynolds). They were examined in a JEM 100B electron microscope. RESULTS
Dogfish erythrocytes remain as flattened, ellipsoid discs after a washing with physiological saline. Upon addition of zinc buffer the red cells swell and become round (Fig. la). Within minutes nuclei move to the periphery of the cell where they contact the inner surface of the plasma membrane, and a significant number of cells (approximately 10-20~o) spontaneously extrude their nuclei leaving a clear plasma membrane bag (Fig. lb). The number of these ghosts is increased by gentle homogenization. The mechanism of nuclear elimination seen in our system is unknown. However, it is a remarkable process, one that has not been seen by us in tissue culture cells manipulated in various ways, including subjection to hypotonic solutions of zinc and other heavy metal ions (Warren & Glick, 1969). Initially, there is swelling and rounding with cells occasionally bursting. When subjected to compression and sudden decompression in a Dounce homogenizer, the cells "pop", which produces a large hole in the plasma membrane through which the nucleus and cytoplasmic contents of the cell are extruded. There is immediate resealing of the membrane and the resultant ghosts are intact bags since they are able to shrink or swell in solutions of appropriate molarity. Virtually all hemoglobin is eliminated, and the final product is colorless. It is not known whether there is transient fusion of nuclear and plasma membrane during extrusion. Recently Reiner and Di Bona have reported briefly on the
extrusion of nuclei from the erythrocytes of toad, frog, amphiuma, turtle and pigeon in hypotonic conditions (approximately 1/4 isoosmotic) (Reiner & Di Bona, 1977). However, we have not seen the enucleation process described in dogfish red cells to take p l a c e ' in duck erythrocytes. Enucleation has also been induced by exposing cells to cytochalasin B, then centrifuging them (Prescott & Kirkpatrick, 1973). In Fig. 2 is seen an electron micrograph of membrane in cross-section. In places, a trilaminar structure can be discerned. Little non-membranous contamination is evident. Analytic results It was found that when preparations of membranes were extracted with chloroform-methanol (2:1) as described previously (Weinstein et al., 1969), 40.5~o of the sample, by charring assay, was lipid (Marsh & Weinstein, 1966). Several preparations of red cells and isolated surface membranes were assayed by the Lowry method (Lowry et al., 1951). It was found that an intact red cell contained 2.56 x 1 0 - 1 ° g protein and an isolated ghost had 4.95 × 10-12g protein (Table 1). The percent of total cell protein found in the ghost was 1.94 while the percent of the ghost that is protein is 53.5. These values are similar to those obtained for the human red cell ghost where the protein-lipid-carbohydrate ratios in two studies were 55:35:10 (Bakerman & Wasemuller, 1967) and 49:44:7 (Rosenberg & Guidotti, 1968). Only traces of D N A and very small amounts of RNA were found in our dogfish red cell membrane preparations. The amino acid composition of the dogfish erythrocyte ghost is seen in Table 2. The amino acid content of the human erythrocyte ghost is also presented for comparison. The amino acids of the plasma membranes of human and dogfish erythrocytes and cultured mouse fibroblasts (Warren & Glick, 1969) were grouped, and the percentage of the total for each type is presented in Table 3. It is evident that all three cell types are remarkably similar in the amino acid composition of their plasma membranes. Carbohydrates In Table 4 the results of analyses of carbohydrates can be seen. The dogfish erythrocyte ghost contained mainly D-mannose and D-galactose, but L-fucose, the hexosamines and sialic acid were also present. All but sialic acid were determined by gas liquid chromatography as their alditol acetates (Lehnhardt & Winzler, 1968). The values reported here are comparable to those for bovine, human, equine and ovine erythrocyte ghosts (Hudson et al., 1975), except that there appears to be considerably more mannose in dogfish erythrocyte ghosts. The relatively high mannose content and the low galactosamine content compared to other types of erythrocyte membrane discussed by Hudson et al. (1975) suggest that dogfish erythrocyte ghosts are relatively rich in "plasma type" glycoproteins (Kraemer, 1971). The distribution of sialic acid in the dogfish red cell was studied. In three experiments it was found that the dogfish ghost contained 76.0-98.2~o of the total sialic acid of the cell (mean 87.6) with a 30-fold increase in concentration over the intact cell (t~mol/mg protein). In five analyses 31.1 + 4.0~o of the
Characterization of erythrocyte membrane of M. canis
Fig. 1(a)
Fig. 1(b)
473
474
L. WARREN et al.
Fig. 1 (c)
Fig. 1 (d) Fig. 1. (a) Washed, intact dogfish erythrocytes. Other cell types have been eliminated leaving only flattened ellipsoidal, nucleated dogfish erythrocytes. (b) Erythrocytes after 5 min at room temperature in hypotonic ZnC12 soln. Note that the cells are rounded and the nuclei have become a flat, uniform grey. Several cells have already lost their nuclei while others are in the process of doing so. Small discrete particles of an u n k n o w n nature are seen in the empty cells. The particles disappear after homogenization and centrifugation. (c) Cells after being cooled were then ruptured in the Dounce homogenizer. Further homogenization is necessary to increase the yield of ghosts, most of which are intact and osmotically active. (d) Final preparation of membranes. In the field are seen several osmotically active ghosts, some of which are irregular in shape. Few fragments and debris but no nuclei are seen. Cells were viewed with a Zeiss phase contrast microscope. Some optical artefact is evident. Magnification × 720.
Characterization of erythrocyte membrane of M. canis
475
Fig. 2. Electron micrograph of purified red blood cell ghosts. Membranes show a typical bilayer structure with very little material adhering to the inside surface of the membrane. Arrow represents the inside of the ghost. Magnification × 40,000.
membrane sialic acid was extractable with chloroform-methanol (2:1); presumably this sialic acid is in gangliosides. Incubation of intact, washed dogfish erythrocytes with excess neuraminidase of V. cholerae in sea water for 3 hr led to the freeing of 50-65~o of the total sialic acid. In one of these experiments 63.5~o of the lipid-soluble sialic acid was released, thus lipid soluble sialic acid was susceptible to neuraminidase as was the protein-bound form. This differs from the lipid-bound sialic acid of fibroblasts which
under our conditions is resistant to neuraminidase (Weinstein et al., 1969; Barton & Rosenberg, 1973). Lipids
The results of detailed lipid analyses are seen in Tables 5-8. The values of these erythrocyte ghosts are compared to those of other species. Approximately 31~o of the lipid was neutral and 66~o was phospholipid; findings in other plasma membranes (cow, dog, rabbit, rat) are similar (Weinstein et al.,
476
L. WARREN et al. Table 1. Overall composition of dogfish erythrocyte ghost A protein composition of intact erythrocyte and ghost of dogfish erythrocyte
Intact erythrocyte Intact ghost
g protein
n
2,56 ___0.20 × 10-1o 4,95 ___0.50 x 10 -12 (1.94~o)
7 14
B Overall composition of dogfish erythrocyte ghost of total cell composition
g per ghost x 10-tz Protein Lipid Carbohydrate (Total)
4.95 3.75 0.55 9.25 g per ghost × 10 t4 trace 2.2
DNA RNA
n
53.5 40.5 6.0 100.0
14 3 3
n = number of experiments performed. Table 2. Amino acid content of erythrocyte plasma membranes Residues/1000 amino acids Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine (half} Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Total residues
Human*
Humant
Dogfish
50.7 24.3 56.4 88.8 52.3 70.5 134 53.5 67.0 87.1 8.2 63.8 19.9 45.2 114 23.0 41.2 N.D. 999.9
47.7 24.0 48.2 81.7 57.2 70.4 121.2 47.8 65.7 80.7 14.3 68.6 24.3 50.6 123.5 25.4 44.8 3.7 999.9
56.5 25.6 44.3 86.8 54.3 76.0 143.7 47.0 79,5 80,8 -t 56,0 24.0 57.9 103.4 25.0 39.1 N.D. 999.9
* Data of Hudson et al. [6]. t Data of Bakerman & Wasemiller [8]. $ Half cystine not found. N.D. = not determined. Table 3. Percentage distribution of amino acids in plasma membranes Erythrocyte Amino acid group
Fibroblast Mouse*
Humant
Humant
Dogfish
Basic Acidic Neutral Proline~ Serine, Threonine Sq Aromatic
14.3 18.7 35.5 6.8 11.9 4.7 8.1
13.1 22.3 37.7 5.4 12.3 2.8 7.4
12.6 21.8 37.0 5.1 12.8 4.0 6.9
12.7 23.0 32.7 4.7 13.0 2.411 6.4
* Membranes isolated by the Zn ion method [20]. t Data of Hudson et al. [6]. $ Data of Bakerman & Wasemiller [8]. § Hydroxyproline not found. II Half cystine not found. • Tryptophan not determined.
Characterization of erythrocyte membrane of M. canis
477
Table 4. Carbohydrate content of dogfish erythrocyte ghosts Sugar
/amoles per ghost x 10 - t ° + S.E.M.
L-fucose D-mannose n-galactose N-acetyI-D-glucosamine N-acetyl-D-galactosamine Sialic acid*
2.03 11.27 19.11 3.42 0.94 5.00
g per ghost x 10 -14
__+0.29 + 0.54 _ 0.54 + 0.49 _ 0.20 _+ 0.99
pg/mg protein n
3.33 20.30 34.42 7.56 2.07 15.45
6.73 41.07 69.56 15.26 4.20 31.21
3 3 3 3 3 6
* Calculated as N-acetylneuraminic acid (MW, 309). n = number of experiments performed.
Table 5. Overall liquid composition of erythrocyte ghost of dogfish* and of other speciest g per ghost x 10 -2 _ S.E.M.
Total Total Total Total
neutral lipid phospholipids other lipids all lipids
~o of total lipid composition
(dogfish)
Dogfish
Cow
Dog
Rabbit
Rat
1.18 + 0.06 2.48 + 0.10 0.09 3.75 _ 0.4
31.56 66.14 2.30 100.0
29.5 69.3 1.2 100.0
28.2 64.4 7.4 100.0
29.9 69.3 0.8 100.0
26.1 73.3 0.6 100.0
Average of three determinations. t Data for other species from G. J. Nelson (1972). *
Table 6. Distribution of neutral lipids of dogfish erythrocyte ghosts of total lipids + S.E.M. Mono- and diglycerides Triglycerides Free fatty acids Methyl esters of fatty acids Cholesterol (total) (Hydrocarbon?) Total
~o of neutral lipids
3.6 +_ 0.4 6.5 + 0.6 2.9 + 0.4 0.8 + 0.1 16.5 _ 1.8 1.3 _ 0.3 31.6~o
11.3 20.5 6.9 2.5 54.7 4.1 100.0~
Average of three determinations.
Table 7. Distribution of phospholipids of dogfish erythrocyte ghosts ~ and other species b ~o of total phospholipids + S.E.M. Dogfish Phosphatidylcholine Lysophosphat idylcholine Phosphatidylethanolamine Lysophosphatidylethanolamine Phosphatidyl serine Phosphatidic acid Plasmalogen: (O-Acyl-alk- 1-enylglycerolphosphorylethanolamine (O-Acyl-alk-l-enylglycerolphosphorylcholine Sphingomyelin Phosphatidyl inositol Other phospholipids Total Average of three determinations. b Data obtained from G. J. Nelson [35].
21.2 0.3 22.8 0.4 16.3 2.4
+ + + _ + +
2.1 0.5 1.7 0.3 0.9 0.7
Dolphin
Seal-harp
18.1 0.5 24.6
32.9 1.7 24.2
16.7 0.1
15.2 0.4
27.8 7.5 4.5 100.0
21.3 2.1 2.1 100.0
2.2 + 0.6 1.2 + 0.2 23.4 ___0.5 6.4 +__ 1.2 3.5 100.0
L. WARREN et al.
478
Table 8. Major fatty acids in lipid fractions of dogfish erythrocyte ghosts
Major fatty acids 10:0
Total neutral lipids
Total sphingomyelin
Total phosphatidyl ethanolamine
Total phosphatidyl choline
0.1 4.0 0.1 4.7 25.2 3.3 0.7 0.3 0.8 37.3 0.2 3.5 0.5 1.2
0.2 2.4 Trace
Total phosphatidic acid
0.9
10:1
1.6
12:0 12:1 14:0 14:1 15:0 (?) 16:0 16:1 16:2 17:0 (?) 18:0 18:1 18:2 18:3 20:0 20:1 20:2 20:3 20:4 20:5 or 22:1 22:0 24:0 24: & 26 polyunsaturated
6.7 1.3 7.5 7.2
Unsaturated fatty acids of, Saturated fatty acids 7/(, Polyunsaturated fatty acids ~o
Trace
16.7
21.2 12.5 1.2
2.8 I 1.2 0.3 0.3 0.6 2.2
3.6 7.1 8.4 60.4 0.4 1.2 0.5 0.6 0.7 0.1 0.8 14.1 2.0
42.9 57.1 17.7
81.0 19.0 72.8
3.2 2.7
1.3 0.2 3.5
20.6 8.9 3.5
29.9 5.2 2.2
15.9 8.3 0.7 20.5
17.1 3.5 0.7 20.2
1.4
Trace
13.8 0.5 0.8 1.2 2.0
1.2 0.8 0.9 9.2 5.7
Trace
62.6 37.4 20.0
51.2 48.8 33.8
36.2 63.8 27.3
Trace 12.l 4.3
Average of three determinations. 1969; Nelson, 1972) (Table 5). There was a tendency for the free fatty acid content to be relatively high, but this was found to be fairly constant in all preparations. The high free fatty acid value is consistent with somewhat higher contents of lysophosphatides, m o n o - and diglycerides in the preparations. P h o s p h a tidic acid values were also somewhat higher in all preparations. Cholesterol (plus cholesterol ester) constituted about 55~o of the neutral lipids, which is not unusual in most species (Nelson, 1972), and the molar cholesterol phosphoslipid ratio is in the commonly found range. There are clearly higher levels of phosphatidyl serine and inositol and lower plasmalogen levels in dogfish t h a n in h u m a n erythrocytes (Mitchell & H a n a h a n , 1966; Van Bruggen, 1971; Cornwell et al., 1968). C o m p a r e d to the dolphin and seal-harp, the phosphatidyl serine and inositol are in a similar range (Nelson, 1972). Fatty acids shorter t h a n 14 appear in the neutral lipids, and in general there was a tendency for the fatty acids to be polyunsaturated, especially in the neutral lipids. In summary, there do not appear to be marked, overall differences in the composition of the surface m e m b r a n e of the nucleated dog fish erythrocyte compared to those of other species. Acknowledgements--We wish to thank Dr C. H. Damsky for performing the electron microscopy, and Dr John C. Hartmann for doing the gas-liquid chromatographic analysis. This work was supported, in part, by USPHS grants CA-13992, CA-14985 and CA-10815 from the National Cancer Institute, grant PRP-28 from the American Cancer
Society and grants from the National Science Foundation. The work of I.W.S. was supported by USPHS research grant AI-13422 from the National Institute of Allergy and Infectious Diseases. Much of this work was done at the Marine Biological Laboratory, Woods Hole, Massachusetts.
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ISOLATION A N D CHARACTERIZATION OF THE ERYTHROCYTE SURFACE MEMBRANE OF THE SMOOTH DOGFISH, M U S T E L U S C A N I S L. WARREN,* C. A. BUCK,* J. L. RABINOWITZt and I. W. SHERMAN+ *The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104; "i'Veterans Administration Hospital, University and Woodland Avenues, Philadelphia, Pennsylvania 19104 and :~Department of Biology, University of California, Riverside, California 92521, U.S.A. (Received 7 August 1978) Al~tract--l. The plasma membrane of the dogfish erythrocyte is characterized. 2. Surface membranes were isolated from dogfish red cells. 3. Cells suspended in a hypotonic zinc chloride buffer (0.5 mM ZnCI2, 5 mM Tris-HCl, pH 8.0) tended to enucleate spontaneously. 4. By gentle homogenization relatively high yields of red cell "ghosts" were formed; these were purified using discontinuous gradients of sucrose and of glycerol solutions. Analyses of protein, carbohydrates and lipids were carried out. 5. There did not appear to be marked overall differences in the composition of the surface membrane of the nucleated dogfish erythrocyte compared to those of other species. INTRODUCTION There are numerous reports on the characterization of plasma membranes from the erythrocytes of vertebrates such as mammals (pig [Lenard, 1970; Hamaguchi & Cleve, 1972; Kobylka et al., 1972; Zwaal & Van Deenan, 19681, sheep [Lenard, 1970; Hamaguchi & Cleve, 1972; Zwaal & Van Deenan, 1968; Cabezas & Seco, 1975; Hudson, et al., 1975,1, horse [Hamaguchi & Cleve, 1972; Cabezas & Seco, 1975; Hudson et al., 1975,1, ox [Hamaguchi & Cleve, 1972; Zwaal & Van Deenan, 19681, rat [Lenard, 1970; Zwaal & Van Deenan, 1968,1, human [Hamaguchi & Cleve, 1972; Kobylka et al., 1972; Zwaal & Van Deenan, 1968; Hudson et al., 1975; Dodge et al., 1963; Bakerman & Wasemuller, 1967], dog [Lenard, 1970; Kobylka et al., 1972], camel [Eitan et al., 1976,1, cat [Kobylka et al., 1972], guinea pig [Hamaguchi & Cleve, 1972,1, rabbit [Hamaguchi & Cleve, 1972,1, goat [Hamaguchi & Cleve, 1972,1, cow [Kobylka et al., 1972; Cabezas & Seco, 1975; Hudson et al., 1975,1); birds (turkey [Cadwell, 1976], goose [Shelton, 1973-1, chicken [Zentgraf et al., 1971; Kleinig et al., 1971; Blanchet, 1974; Jackson, 1975], pigeon [Puchwein et al., 1974,1); and frogs (Mukherjee et al., 1976). The isolation of erythrocyte membranes from the lamprey has been reported (Asai et al., 1976). This communication presents a simple, rapid and effective method for the isolation of purified erythrocyte membranes from the elasmobranch Mustelus canis (smooth dogfish). In the technique employed, yields of pure membranes are high and organelle contamination is minimal. The membranes obtained by this method were analyzed for protein, carbohydrates and lipids. MATERIALS AND
METHODS
Preparation of dogfish red blood cell plasma membranes Smooth dogfish were obtained from the Supply Department, Marine Biological Laboratory, Woods Hole, Mass.
Blood was taken from the subcaudal vein of dogfish with a glass or plastic syringe fitted with an 18-gauge needle. The volume removed varied with the size of the animal, 100ml being the maximum taken from the largest (1 m in length) animals. Coagulation of blood was prevented by the addition of 1/10 volume of 0.1 M sodium citrate to the syringe. Twelve and a half ml of whole blood was layered over a 30-ml cushion of 30% (w/v) sucrose solution and centrifuged at 500 g for 10 min; plasma and leukocytes in the upper part of the tube were removed by aspiration. The pellet of red cells was washed twice in 0.9% (w/v) NaC1 (5min, 750g); the cells were then resuspended in an equal volume of 0.9% NaCI. To 10 ml of red cell suspension were added 50mg of solid NaHSO3, which served to inhibit proteases (Panyim & Chalkley, 1969) as well as to make the plasma membrane tougher. The contents of the tube were mixed rapidly and thoroughly. Thirty ml of "zinc buffer" were then added to 10ml of bisulfitetreated red cells in a large Dounce homogenizing tube (40ml, Kontes Glass, Co., Vineland, N.J.) (Warren & Glick, 1969). Zinc buffer contained 0.5 mM ZnCI2 and 5 mM Tris-HC1, pH 8.0. After standing at room temperature for 10 rain the homogenizing tube was transferred to an ice bucket and allowed to cool to 5°C. The cells were then broken with 20-40 strokes of an A pestle. Cell breakage and enucleation were monitored by phase contrast microscopy. Homogenization was stopped when 90% of the cells were broken. Twenty ml of the homogenate were layered over a double sucrose cushion (7.5 ml of 50~,/, (w/v) sucrose solution made in Tris-saline (0.125 M NaCI, 5mM KCI, 1.1 mM CaCI2, 25mM Tris-HCl, pH 7.6), 8ml of 25% (w/v) sucrose solution in Tris-saline) in a 40-ml centrifuge tube. The tube was centrifuged for 1 hr at 2500 rpm (1400 g) in a refrigerated International Centrifuge (Model PR-2). A slightly pink opalescent layer was seen at the interface between the 25 and 50% sucrose layers. This was collected with a 25-ml syringe fitted with a No. 18 needle that had its terminal 4 mm bent so that the bevel faced upward. The opalescent layer was diluted with an equal volume of Tris-saline buffer. A discontinuous glycerol gradient was made in a 40 ml centrifuge tube consisting of 10 ml of 40~o glycerol (v/v), 7.5 ml of 50% and 7.5 ml of 70% glycerol all made in Trissaline buffer. The tube was centrifuged in a PR-2 centrifuge for 2 hr at 3000 rpm (2000 g) at 4°C. A colorless opalescent 471
472
L. WARRENet al.
material just above the 70~o glycerol layer was collected with a syringe, diluted with 30 ml of Tris-saline buffer and centrifuged for 1 hr at 2500 rpm (1400 g) at 4°C in the PR-2 centrifuge. A pale white opalescent pellet was collected. The final yield of purified, intact "ghosts" (red cell plasma membranes) was usually about 25~0 of the original number of red cells as measured by hemocytometer counts. All procedures were carried out at 4°C unless stated otherwise. Purification procedures were monitored by phase contrast microscopy. All chemicals used were of commercial reagent grade. Neuraminidase (V. cholerae) was purchased from Calbiochem, La Jolla, California. Sialic acid was measured by the thiobarbituric acid assay (Warren, 1959) after hydrolysis with 0.1 N H2SO4 at 80~C for 1 hr. All other sugars were measured as their alditol acetates by gas-liquid chromatography (Lehnhardt & Winzler, 1968). Lipids were separated and analyzed by methods previously described in detail (Weinstein et al., 1969). DNA and RNA were analyzed by the methods of Dische (1930) and Mejbaum (1939), respectively. Amino acid analysis was carried out using a Beckmann amino acid analyzer by the method of Moore & Stein 11945)after 24hr hydrolysis at 105"C in 6 N HC1 in sealed, evacuated ampules. Analyses were carried out by Mr. R. Hyde at the Marine Biological Laboratory, Woods Hole, Massachusetts. Electron microscopy of purified ghosts Small pellets of dogfish red cell ghosts were fixed with 2~o (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, followed by osmium tetroxide in 0.1M cacodylate. Samples were dehydrated in ethanol and propylene oxide and embedded in Epon. Sections were cut on an MT-2 microtome (Sorvall-Dupont) and stained with a saturated solution of uranyl acetate in 50,50 ethanol and with lead citrate (Reynolds). They were examined in a JEM 100B electron microscope. RESULTS
Dogfish erythrocytes remain as flattened, ellipsoid discs after a washing with physiological saline. Upon addition of zinc buffer the red cells swell and become round (Fig. la). Within minutes nuclei move to the periphery of the cell where they contact the inner surface of the plasma membrane, and a significant number of cells (approximately 10-20~o) spontaneously extrude their nuclei leaving a clear plasma membrane bag (Fig. lb). The number of these ghosts is increased by gentle homogenization. The mechanism of nuclear elimination seen in our system is unknown. However, it is a remarkable process, one that has not been seen by us in tissue culture cells manipulated in various ways, including subjection to hypotonic solutions of zinc and other heavy metal ions (Warren & Glick, 1969). Initially, there is swelling and rounding with cells occasionally bursting. When subjected to compression and sudden decompression in a Dounce homogenizer, the cells "pop", which produces a large hole in the plasma membrane through which the nucleus and cytoplasmic contents of the cell are extruded. There is immediate resealing of the membrane and the resultant ghosts are intact bags since they are able to shrink or swell in solutions of appropriate molarity. Virtually all hemoglobin is eliminated, and the final product is colorless. It is not known whether there is transient fusion of nuclear and plasma membrane during extrusion. Recently Reiner and Di Bona have reported briefly on the
extrusion of nuclei from the erythrocytes of toad, frog, amphiuma, turtle and pigeon in hypotonic conditions (approximately 1/4 isoosmotic) (Reiner & Di Bona, 1977). However, we have not seen the enucleation process described in dogfish red cells to take p l a c e ' in duck erythrocytes. Enucleation has also been induced by exposing cells to cytochalasin B, then centrifuging them (Prescott & Kirkpatrick, 1973). In Fig. 2 is seen an electron micrograph of membrane in cross-section. In places, a trilaminar structure can be discerned. Little non-membranous contamination is evident. Analytic results It was found that when preparations of membranes were extracted with chloroform-methanol (2:1) as described previously (Weinstein et al., 1969), 40.5~o of the sample, by charring assay, was lipid (Marsh & Weinstein, 1966). Several preparations of red cells and isolated surface membranes were assayed by the Lowry method (Lowry et al., 1951). It was found that an intact red cell contained 2.56 x 1 0 - 1 ° g protein and an isolated ghost had 4.95 × 10-12g protein (Table 1). The percent of total cell protein found in the ghost was 1.94 while the percent of the ghost that is protein is 53.5. These values are similar to those obtained for the human red cell ghost where the protein-lipid-carbohydrate ratios in two studies were 55:35:10 (Bakerman & Wasemuller, 1967) and 49:44:7 (Rosenberg & Guidotti, 1968). Only traces of D N A and very small amounts of RNA were found in our dogfish red cell membrane preparations. The amino acid composition of the dogfish erythrocyte ghost is seen in Table 2. The amino acid content of the human erythrocyte ghost is also presented for comparison. The amino acids of the plasma membranes of human and dogfish erythrocytes and cultured mouse fibroblasts (Warren & Glick, 1969) were grouped, and the percentage of the total for each type is presented in Table 3. It is evident that all three cell types are remarkably similar in the amino acid composition of their plasma membranes. Carbohydrates In Table 4 the results of analyses of carbohydrates can be seen. The dogfish erythrocyte ghost contained mainly D-mannose and D-galactose, but L-fucose, the hexosamines and sialic acid were also present. All but sialic acid were determined by gas liquid chromatography as their alditol acetates (Lehnhardt & Winzler, 1968). The values reported here are comparable to those for bovine, human, equine and ovine erythrocyte ghosts (Hudson et al., 1975), except that there appears to be considerably more mannose in dogfish erythrocyte ghosts. The relatively high mannose content and the low galactosamine content compared to other types of erythrocyte membrane discussed by Hudson et al. (1975) suggest that dogfish erythrocyte ghosts are relatively rich in "plasma type" glycoproteins (Kraemer, 1971). The distribution of sialic acid in the dogfish red cell was studied. In three experiments it was found that the dogfish ghost contained 76.0-98.2~o of the total sialic acid of the cell (mean 87.6) with a 30-fold increase in concentration over the intact cell (t~mol/mg protein). In five analyses 31.1 + 4.0~o of the
Characterization of erythrocyte membrane of M. canis
Fig. 1(a)
Fig. 1(b)
473
474
L. WARREN et al.
Fig. 1 (c)
Fig. 1 (d) Fig. 1. (a) Washed, intact dogfish erythrocytes. Other cell types have been eliminated leaving only flattened ellipsoidal, nucleated dogfish erythrocytes. (b) Erythrocytes after 5 min at room temperature in hypotonic ZnC12 soln. Note that the cells are rounded and the nuclei have become a flat, uniform grey. Several cells have already lost their nuclei while others are in the process of doing so. Small discrete particles of an u n k n o w n nature are seen in the empty cells. The particles disappear after homogenization and centrifugation. (c) Cells after being cooled were then ruptured in the Dounce homogenizer. Further homogenization is necessary to increase the yield of ghosts, most of which are intact and osmotically active. (d) Final preparation of membranes. In the field are seen several osmotically active ghosts, some of which are irregular in shape. Few fragments and debris but no nuclei are seen. Cells were viewed with a Zeiss phase contrast microscope. Some optical artefact is evident. Magnification × 720.
Characterization of erythrocyte membrane of M. canis
475
Fig. 2. Electron micrograph of purified red blood cell ghosts. Membranes show a typical bilayer structure with very little material adhering to the inside surface of the membrane. Arrow represents the inside of the ghost. Magnification × 40,000.
membrane sialic acid was extractable with chloroform-methanol (2:1); presumably this sialic acid is in gangliosides. Incubation of intact, washed dogfish erythrocytes with excess neuraminidase of V. cholerae in sea water for 3 hr led to the freeing of 50-65~o of the total sialic acid. In one of these experiments 63.5~o of the lipid-soluble sialic acid was released, thus lipid soluble sialic acid was susceptible to neuraminidase as was the protein-bound form. This differs from the lipid-bound sialic acid of fibroblasts which
under our conditions is resistant to neuraminidase (Weinstein et al., 1969; Barton & Rosenberg, 1973). Lipids
The results of detailed lipid analyses are seen in Tables 5-8. The values of these erythrocyte ghosts are compared to those of other species. Approximately 31~o of the lipid was neutral and 66~o was phospholipid; findings in other plasma membranes (cow, dog, rabbit, rat) are similar (Weinstein et al.,
476
L. WARREN et al. Table 1. Overall composition of dogfish erythrocyte ghost A protein composition of intact erythrocyte and ghost of dogfish erythrocyte
Intact erythrocyte Intact ghost
g protein
n
2,56 ___0.20 × 10-1o 4,95 ___0.50 x 10 -12 (1.94~o)
7 14
B Overall composition of dogfish erythrocyte ghost of total cell composition
g per ghost x 10-tz Protein Lipid Carbohydrate (Total)
4.95 3.75 0.55 9.25 g per ghost × 10 t4 trace 2.2
DNA RNA
n
53.5 40.5 6.0 100.0
14 3 3
n = number of experiments performed. Table 2. Amino acid content of erythrocyte plasma membranes Residues/1000 amino acids Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine (half} Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Total residues
Human*
Humant
Dogfish
50.7 24.3 56.4 88.8 52.3 70.5 134 53.5 67.0 87.1 8.2 63.8 19.9 45.2 114 23.0 41.2 N.D. 999.9
47.7 24.0 48.2 81.7 57.2 70.4 121.2 47.8 65.7 80.7 14.3 68.6 24.3 50.6 123.5 25.4 44.8 3.7 999.9
56.5 25.6 44.3 86.8 54.3 76.0 143.7 47.0 79,5 80,8 -t 56,0 24.0 57.9 103.4 25.0 39.1 N.D. 999.9
* Data of Hudson et al. [6]. t Data of Bakerman & Wasemiller [8]. $ Half cystine not found. N.D. = not determined. Table 3. Percentage distribution of amino acids in plasma membranes Erythrocyte Amino acid group
Fibroblast Mouse*
Humant
Humant
Dogfish
Basic Acidic Neutral Proline~ Serine, Threonine Sq Aromatic
14.3 18.7 35.5 6.8 11.9 4.7 8.1
13.1 22.3 37.7 5.4 12.3 2.8 7.4
12.6 21.8 37.0 5.1 12.8 4.0 6.9
12.7 23.0 32.7 4.7 13.0 2.411 6.4
* Membranes isolated by the Zn ion method [20]. t Data of Hudson et al. [6]. $ Data of Bakerman & Wasemiller [8]. § Hydroxyproline not found. II Half cystine not found. • Tryptophan not determined.
Characterization of erythrocyte membrane of M. canis
477
Table 4. Carbohydrate content of dogfish erythrocyte ghosts Sugar
/amoles per ghost x 10 - t ° + S.E.M.
L-fucose D-mannose n-galactose N-acetyI-D-glucosamine N-acetyl-D-galactosamine Sialic acid*
2.03 11.27 19.11 3.42 0.94 5.00
g per ghost x 10 -14
__+0.29 + 0.54 _ 0.54 + 0.49 _ 0.20 _+ 0.99
pg/mg protein n
3.33 20.30 34.42 7.56 2.07 15.45
6.73 41.07 69.56 15.26 4.20 31.21
3 3 3 3 3 6
* Calculated as N-acetylneuraminic acid (MW, 309). n = number of experiments performed.
Table 5. Overall liquid composition of erythrocyte ghost of dogfish* and of other speciest g per ghost x 10 -2 _ S.E.M.
Total Total Total Total
neutral lipid phospholipids other lipids all lipids
~o of total lipid composition
(dogfish)
Dogfish
Cow
Dog
Rabbit
Rat
1.18 + 0.06 2.48 + 0.10 0.09 3.75 _ 0.4
31.56 66.14 2.30 100.0
29.5 69.3 1.2 100.0
28.2 64.4 7.4 100.0
29.9 69.3 0.8 100.0
26.1 73.3 0.6 100.0
Average of three determinations. t Data for other species from G. J. Nelson (1972). *
Table 6. Distribution of neutral lipids of dogfish erythrocyte ghosts of total lipids + S.E.M. Mono- and diglycerides Triglycerides Free fatty acids Methyl esters of fatty acids Cholesterol (total) (Hydrocarbon?) Total
~o of neutral lipids
3.6 +_ 0.4 6.5 + 0.6 2.9 + 0.4 0.8 + 0.1 16.5 _ 1.8 1.3 _ 0.3 31.6~o
11.3 20.5 6.9 2.5 54.7 4.1 100.0~
Average of three determinations.
Table 7. Distribution of phospholipids of dogfish erythrocyte ghosts ~ and other species b ~o of total phospholipids + S.E.M. Dogfish Phosphatidylcholine Lysophosphat idylcholine Phosphatidylethanolamine Lysophosphatidylethanolamine Phosphatidyl serine Phosphatidic acid Plasmalogen: (O-Acyl-alk- 1-enylglycerolphosphorylethanolamine (O-Acyl-alk-l-enylglycerolphosphorylcholine Sphingomyelin Phosphatidyl inositol Other phospholipids Total Average of three determinations. b Data obtained from G. J. Nelson [35].
21.2 0.3 22.8 0.4 16.3 2.4
+ + + _ + +
2.1 0.5 1.7 0.3 0.9 0.7
Dolphin
Seal-harp
18.1 0.5 24.6
32.9 1.7 24.2
16.7 0.1
15.2 0.4
27.8 7.5 4.5 100.0
21.3 2.1 2.1 100.0
2.2 + 0.6 1.2 + 0.2 23.4 ___0.5 6.4 +__ 1.2 3.5 100.0
L. WARREN et al.
478
Table 8. Major fatty acids in lipid fractions of dogfish erythrocyte ghosts
Major fatty acids 10:0
Total neutral lipids
Total sphingomyelin
Total phosphatidyl ethanolamine
Total phosphatidyl choline
0.1 4.0 0.1 4.7 25.2 3.3 0.7 0.3 0.8 37.3 0.2 3.5 0.5 1.2
0.2 2.4 Trace
Total phosphatidic acid
0.9
10:1
1.6
12:0 12:1 14:0 14:1 15:0 (?) 16:0 16:1 16:2 17:0 (?) 18:0 18:1 18:2 18:3 20:0 20:1 20:2 20:3 20:4 20:5 or 22:1 22:0 24:0 24: & 26 polyunsaturated
6.7 1.3 7.5 7.2
Unsaturated fatty acids of, Saturated fatty acids 7/(, Polyunsaturated fatty acids ~o
Trace
16.7
21.2 12.5 1.2
2.8 I 1.2 0.3 0.3 0.6 2.2
3.6 7.1 8.4 60.4 0.4 1.2 0.5 0.6 0.7 0.1 0.8 14.1 2.0
42.9 57.1 17.7
81.0 19.0 72.8
3.2 2.7
1.3 0.2 3.5
20.6 8.9 3.5
29.9 5.2 2.2
15.9 8.3 0.7 20.5
17.1 3.5 0.7 20.2
1.4
Trace
13.8 0.5 0.8 1.2 2.0
1.2 0.8 0.9 9.2 5.7
Trace
62.6 37.4 20.0
51.2 48.8 33.8
36.2 63.8 27.3
Trace 12.l 4.3
Average of three determinations. 1969; Nelson, 1972) (Table 5). There was a tendency for the free fatty acid content to be relatively high, but this was found to be fairly constant in all preparations. The high free fatty acid value is consistent with somewhat higher contents of lysophosphatides, m o n o - and diglycerides in the preparations. P h o s p h a tidic acid values were also somewhat higher in all preparations. Cholesterol (plus cholesterol ester) constituted about 55~o of the neutral lipids, which is not unusual in most species (Nelson, 1972), and the molar cholesterol phosphoslipid ratio is in the commonly found range. There are clearly higher levels of phosphatidyl serine and inositol and lower plasmalogen levels in dogfish t h a n in h u m a n erythrocytes (Mitchell & H a n a h a n , 1966; Van Bruggen, 1971; Cornwell et al., 1968). C o m p a r e d to the dolphin and seal-harp, the phosphatidyl serine and inositol are in a similar range (Nelson, 1972). Fatty acids shorter t h a n 14 appear in the neutral lipids, and in general there was a tendency for the fatty acids to be polyunsaturated, especially in the neutral lipids. In summary, there do not appear to be marked, overall differences in the composition of the surface m e m b r a n e of the nucleated dog fish erythrocyte compared to those of other species. Acknowledgements--We wish to thank Dr C. H. Damsky for performing the electron microscopy, and Dr John C. Hartmann for doing the gas-liquid chromatographic analysis. This work was supported, in part, by USPHS grants CA-13992, CA-14985 and CA-10815 from the National Cancer Institute, grant PRP-28 from the American Cancer
Society and grants from the National Science Foundation. The work of I.W.S. was supported by USPHS research grant AI-13422 from the National Institute of Allergy and Infectious Diseases. Much of this work was done at the Marine Biological Laboratory, Woods Hole, Massachusetts.
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