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Solubilization and comparative study of the erythrocyte membrane proteins and glycoproteins from various mammalian species
Comp. Biochem. PhysioL, 1975, Vol. 51B,pp. 243 to 245. Pergamon Press. Printed in Great Britain
SOLUBILIZAT!ON AND COMPARATIVE STUDY OF THE ERYTHROCYTE MEMBRANE PROTEINS AND GLYCOPROTEINS FROM VARIOUS MAMMALIAN SPECIES J. A. ~ a s z ~ ANY A. Seco Department of Biochemistry, Faculty of Sciences, University of Salamanca, Salamanca, Spain (Received 14 March 1974)
Al~raet-1. Erythrocyte membranes from the horse (Equus caballus L.), mule (Equus cabal/us x Equu asinus), donkey (Equus us/n~ L.), cow (Bos taurus L), pig (Sus scrofa L.) and sheep (Ovis aries L.) were solubflized by four different procedures. 2. These procedures are: (A) an aqueous solution of 0.1 N NaOH at pH 8.0; (B) acetic acid-ureaphenol-mm'captoethanol; (C) EDTA-mercaptoethanol-SDS with heating; and ('13)SDS--phesphate-mercaptoethanol-glycerol. 3. The gel acrylamide electrophoretic patterns obtained with the proteins and glycoproteins of the erythrocyte membranes from the above-mentioned species show some similarities as well as some differences. 4. These similarities are especiallymarked, in both protein andglycoprotein electrophoreticpatterns, for the horse, mule and donkey. 5. The molecular weight of the proteins ranged between 250,000 and 22,000. 6. The best resolution in the separation of proteins by gel acrylamide was achieved with procedure
(c).
7. The same sialic acid, nearly 100%N-glycolyineuramlnicacid, was found in the erythrocyte membrane of the horse, mule and donkey; this acid as being predominant, but also N-acetylneuraminicacid, was determined in erythrocy~ from the cow, pig and sheep.
INTRODUCTION
ONE approach for the study of proteins and glycoproteins of erythrocyte membranes is to solubilize the membrane as much as poss~le, but the solubility of the proteins of the cell membrane is low. Mechanical means and very different agents (or~,anic solvents, detergents, denaturants, salts, etc.) havebeen widely used for the preparation of soluble erythrocyte membrane proteins; for example, butanol, sodium dodecyl sulfate (SDS), formic acid, acetic acid, NaI, mersalyl, pyridine, EDTA, (CHa)NBr, hexafluoroaeetone, guanidine hydrochloride, picolines, ~fidine, Triton X-100, ATP-mercaptoethanol, urea, cetyltrimethyl ammonium bromide-guanidine hydrochloride, phenol-acetic acid-water together with several others in association (Razin, 1972; Cabezas, unpublished data). However, ~they have often been employed in such conditions that might degrade proteim or glycoproteins to polypeptido chains. Actually, the data available on the solubility of membrane proteins are controversial. On the other hand, it seems desirable to compare the data concerning the proteins and glycoproteins of the erythrocyte mel~branes from several species because the/r functional properties are considered to be related with their composition. Electropboretic
methods, especially polyacrylamide gel electrophoresis, offer superior advantages for this comparison. Zwaal & van Deenen (1968), Carraway & Kobylka (1970), Lenard (1970), Gomperts et al. (1971), Hamaguchi & Cleve 0972) and KobYlka et a/. (1972) have compared the electrophoretic patterns of the solubilized e r y t h r o c ~ membrane proteins from human (Homo sapiens), monkey, horse, cow, pig, sheep, goat (Capra pyrenaica victoriae), dog (Canis familiaris L.), cat (Fells catus L.), rabbit (Oryctolagus euniculus L.) and guinea pig (Cav/a cobaya); but several variables appear responsible for conflicting reports in some cases. In the present work, we have employed four procedures for the soinbfiization of proteins and glycoproteins of erytlirocyte ~ from some mammalian species not previously studied (mule and donkey), and als0for the horse, cow, pig and sheep. Also, our approach differs from that of other workers since we have employed the method of Eylar et al. (196.2) for the preparation of erythrocyte man~ and ~ ~ we have tried to soiubilize the maximum qt~ntity of all erythrocyte membrane pmteim, and not to extract one specific protein. F ~ , we have determined the type and content o f sialic acid in the erythrocyte glycoproteins from the above-mentioned species to
243
244
J . A . C,~mez~ AND A. SEco
compare phylogenetic differences among closely related species. MATERIALS AND METHODS Erythrocyte m e m b r a ~ were p~pared essentially by the method of Eylar et al. (1962). The blood was obtained at a local slaughter-house and collected into 3.8~ sodium citrate. After centrifugation, the erythrocytes were washed three times in 10 eel. of isotonic buffer, pH 7.2 (0.135 M sodium chloride and 0.02 M sodium phosphate). The washed erythrocytes were slowly diluted to 10 eel. with distilled water and the pH adjusted between 5 and 6 with 0.1 N HCI. After standing for 5 hr, the supernatant was decanted. The stroma was carefully washed (this treatment being repeated at least five times) until the supernatant fluid showed no visual trace of hemoglobin. All the steps of this procedure were carried out at a temperature of 0-4°C. Ghosts were dried at reduced pressure and stored at 0°C. For the comparative study of proteins, pooled samples were etectrophoresed in polyacrylamide gel by four different procedures: (A) Erythrocyte membranes were solubilized in an aequous solution adjusted to pH 8.0 with 0.1N NaOH (2rag of the sample for 1 ml of solution). Gels were prepared and run essentially according to the standard procedures of Davis (1964) and Ornstein (1964). Acrylamide (7.5~) was used for preparation of the gels. (B) The procedure of Ray & Marineti (1971) (which is a modification of Takayama's method designed for the electrophoresls of plasma membranes). The membranes were dissolved in a mixture of acetic acid-urea-phenolmercaptoethanol; the solvent mixture for running the gel was composed of 6~ acrylamide, methylenebisacrylamide, urea, acetic acid, ammonium persulfate and tetraethylethylene diamino. A mixture of aniline blue black and coomassie brilliant blue in acetic acid was used for staining. (C) The method of Lenard (1970) was used. Membranes were disaggregated by dialysis into EDTAmercaptoethanol (pH 7~)) for a period of 2 days; after the addition of SDS to a final concentration of 2-3~ and heating for 3 rain at 100°C, the elec~rophoresis was run according to Shapiro et al. (1967), except for the staining, which was done by aniline black instead of coomassie blue. (D) Here the procedure of Carraway & Kobylka (1970) was followed. The membranes were dissolved in SDS-phosphate buffer (pH 7.8) containing mercaptoethanol and glycerol; electrophoresiswas run as indicated in ( C ) . . For the electrophoretic study of glycoproteins, membranes were solubilized according to Lenard (1970) and staining was d o ~ with periodic acid--Sch~. The molecular weights of proteins, sepmated to procedures 03) and (C), wen~.estimated with the u~e of trypsin (24,000), glyadin (27j)00), o ~ (44,000), seralbumin (65,000) and ~gl0bulins (160,000) as standard proteins. In some cases, the lipids of the erythrocyte raembranes were removed by a chloroform-methanol (2 : 1, v/v) extraction procedure, for 4 hr, several times. Hydrolysis for sialic acid was performed with 250 mg of erythroeyte ghosts (dry weight) in 10ml of 0-1N HISO, at 80°C, for 1 hr; the hydrolysis was repeated three or four times. Ba(OH)t was added Until the pH was 5.1, and the przcipitate was eliminated by centrifusation. After purification (Cabezas et al., 1964), sialic acids were identified by descending paper (Schieicher & Schfill
2043b) chromatography, with the use of the following solvents: (i) n-butanol-acetic acid-water ( 4 : 1 : 5 ) (72 hr); (ii) n-propanol-0.1N HCI ( 1 : 2 : 1) ( 4 8rh) ; (iii) n-~tanol-pyridine-0.1 N HC1 (5 : 3 : 2) (96 hr) (v/v). Sialic acids were located on paper with p-dimethylaminobenzaldehide (modified Ehrllch reagent) (Bezkorovalny, 1963). For the estimation of the ratio of each sialic acid, the respective zone of paper was eluted with a mixture of methanol-acetic acid (2 : 1) and the eluates read to 483 rim. In some cases, the Eegriwe test (Falllard & Cabezas, 1963) was used to determine N-glycolylneuraminic acid. The hydrolysis for hexoses was carried out with 1 N HC1 at 100°C for 4 hr; and for hexosamines, with 3 N HCI at 100°C for 4 hr. Oses and osamines were located on paper by a AgNes solution. For the quantitative determination of sailic acids, the modified resorcinol procedure (Cabezas et al., 1964) was applied Oses (with the use of galactosc-manncee, 1 : 1, as standard), bexosamines (with a standard of glucosamine chlorhydrate) and proteins were determined as previously reported (Cabezas et al., 1964, 1972). RESULTS AND DISCUSSION The best resolution in the proteins' electrophoretic patterns, obtained with the use of gel acrylamide, has been achieved with procedure (C) (Fig. 1). When procedures (A), (B) or (D) were employed the results were of less resolution and were difficult to interpret. The molecular weights for erythrocyte membrane proteins were estimated as ranging between 250,000 and 22,000. Figure 2 shows the pattern of glycoproteins, obtained essentially by procedure (C). As expected, the highest similarity for proteins and glycoproteins was observed between the horse, mule and donkey s a m p l e s ; the patterns among other species show more marked differences. A parallel result was previously found for serum proteins and glycoproteins of all the above-mentioned species (Cabezas et al., 1964, 1972). Some variations in the results reported by different authors could arise from variations in the preparation of the erythrocyte membranes; also, by the use o f very different agents for solubilization of the proteins and glycoproteins; and, finally, by the utilization o f several modalities of gel electrophoresis. On the other hand, it is known that the charge o f sialic acid play an important role in the mobility of the glycoproteins, and that this acid is located at the outer surface o f the cell. We have liberated all the siatic acid f r o m tbe erythrocyte membranes after two or three very mild hydrolyses; the concentration o f the released sialic acid decreases from the first to the second and third hydrolyses. The type and content of the sialic acids found in the above-mentioned species can be seen in Table 1. The same type of sialic acid, nearly 100~o N-glycolylneuraminic acid, has been detected in the horse, mule and donkey; in the cow and sheep, about a 80% o f sialic acids are N-glycolylneuraminic acid, with the remainder being N-acetylneuraminic acid; finally, in the pig there exist these acids and one other acid, probably a
Fig: 1. Acrylamide gel electrophoretic patterns of erythrocyte membrane proteins. The membranes were solubilized and the electrophoresis was carded out as explained in the text (procedure C). I, Horse; 2, mule; 3, donkey; 4, cow; 5, pig; 6, sheep. Fig. 2. Acrylamide gel electrophoretic patterns of erythrocyte membrane glyeoproteins. The membranes were solubilized and the electrophoresis was carried out as explained in the text. 1, Horse; 2, mule; 3, donkey; 4, cow; 5, pig; 6, sheep.
SOLUBILIZAT!ON AND COMPARATIVE STUDY OF THE ERYTHROCYTE MEMBRANE PROTEINS AND GLYCOPROTEINS FROM VARIOUS MAMMALIAN SPECIES J. A. ~ a s z ~ ANY A. Seco Department of Biochemistry, Faculty of Sciences, University of Salamanca, Salamanca, Spain (Received 14 March 1974)
Al~raet-1. Erythrocyte membranes from the horse (Equus caballus L.), mule (Equus cabal/us x Equu asinus), donkey (Equus us/n~ L.), cow (Bos taurus L), pig (Sus scrofa L.) and sheep (Ovis aries L.) were solubflized by four different procedures. 2. These procedures are: (A) an aqueous solution of 0.1 N NaOH at pH 8.0; (B) acetic acid-ureaphenol-mm'captoethanol; (C) EDTA-mercaptoethanol-SDS with heating; and ('13)SDS--phesphate-mercaptoethanol-glycerol. 3. The gel acrylamide electrophoretic patterns obtained with the proteins and glycoproteins of the erythrocyte membranes from the above-mentioned species show some similarities as well as some differences. 4. These similarities are especiallymarked, in both protein andglycoprotein electrophoreticpatterns, for the horse, mule and donkey. 5. The molecular weight of the proteins ranged between 250,000 and 22,000. 6. The best resolution in the separation of proteins by gel acrylamide was achieved with procedure
(c).
7. The same sialic acid, nearly 100%N-glycolyineuramlnicacid, was found in the erythrocyte membrane of the horse, mule and donkey; this acid as being predominant, but also N-acetylneuraminicacid, was determined in erythrocy~ from the cow, pig and sheep.
INTRODUCTION
ONE approach for the study of proteins and glycoproteins of erythrocyte membranes is to solubilize the membrane as much as poss~le, but the solubility of the proteins of the cell membrane is low. Mechanical means and very different agents (or~,anic solvents, detergents, denaturants, salts, etc.) havebeen widely used for the preparation of soluble erythrocyte membrane proteins; for example, butanol, sodium dodecyl sulfate (SDS), formic acid, acetic acid, NaI, mersalyl, pyridine, EDTA, (CHa)NBr, hexafluoroaeetone, guanidine hydrochloride, picolines, ~fidine, Triton X-100, ATP-mercaptoethanol, urea, cetyltrimethyl ammonium bromide-guanidine hydrochloride, phenol-acetic acid-water together with several others in association (Razin, 1972; Cabezas, unpublished data). However, ~they have often been employed in such conditions that might degrade proteim or glycoproteins to polypeptido chains. Actually, the data available on the solubility of membrane proteins are controversial. On the other hand, it seems desirable to compare the data concerning the proteins and glycoproteins of the erythrocyte mel~branes from several species because the/r functional properties are considered to be related with their composition. Electropboretic
methods, especially polyacrylamide gel electrophoresis, offer superior advantages for this comparison. Zwaal & van Deenen (1968), Carraway & Kobylka (1970), Lenard (1970), Gomperts et al. (1971), Hamaguchi & Cleve 0972) and KobYlka et a/. (1972) have compared the electrophoretic patterns of the solubilized e r y t h r o c ~ membrane proteins from human (Homo sapiens), monkey, horse, cow, pig, sheep, goat (Capra pyrenaica victoriae), dog (Canis familiaris L.), cat (Fells catus L.), rabbit (Oryctolagus euniculus L.) and guinea pig (Cav/a cobaya); but several variables appear responsible for conflicting reports in some cases. In the present work, we have employed four procedures for the soinbfiization of proteins and glycoproteins of erytlirocyte ~ from some mammalian species not previously studied (mule and donkey), and als0for the horse, cow, pig and sheep. Also, our approach differs from that of other workers since we have employed the method of Eylar et al. (196.2) for the preparation of erythrocyte man~ and ~ ~ we have tried to soiubilize the maximum qt~ntity of all erythrocyte membrane pmteim, and not to extract one specific protein. F ~ , we have determined the type and content o f sialic acid in the erythrocyte glycoproteins from the above-mentioned species to
243
244
J . A . C,~mez~ AND A. SEco
compare phylogenetic differences among closely related species. MATERIALS AND METHODS Erythrocyte m e m b r a ~ were p~pared essentially by the method of Eylar et al. (1962). The blood was obtained at a local slaughter-house and collected into 3.8~ sodium citrate. After centrifugation, the erythrocytes were washed three times in 10 eel. of isotonic buffer, pH 7.2 (0.135 M sodium chloride and 0.02 M sodium phosphate). The washed erythrocytes were slowly diluted to 10 eel. with distilled water and the pH adjusted between 5 and 6 with 0.1 N HCI. After standing for 5 hr, the supernatant was decanted. The stroma was carefully washed (this treatment being repeated at least five times) until the supernatant fluid showed no visual trace of hemoglobin. All the steps of this procedure were carried out at a temperature of 0-4°C. Ghosts were dried at reduced pressure and stored at 0°C. For the comparative study of proteins, pooled samples were etectrophoresed in polyacrylamide gel by four different procedures: (A) Erythrocyte membranes were solubilized in an aequous solution adjusted to pH 8.0 with 0.1N NaOH (2rag of the sample for 1 ml of solution). Gels were prepared and run essentially according to the standard procedures of Davis (1964) and Ornstein (1964). Acrylamide (7.5~) was used for preparation of the gels. (B) The procedure of Ray & Marineti (1971) (which is a modification of Takayama's method designed for the electrophoresls of plasma membranes). The membranes were dissolved in a mixture of acetic acid-urea-phenolmercaptoethanol; the solvent mixture for running the gel was composed of 6~ acrylamide, methylenebisacrylamide, urea, acetic acid, ammonium persulfate and tetraethylethylene diamino. A mixture of aniline blue black and coomassie brilliant blue in acetic acid was used for staining. (C) The method of Lenard (1970) was used. Membranes were disaggregated by dialysis into EDTAmercaptoethanol (pH 7~)) for a period of 2 days; after the addition of SDS to a final concentration of 2-3~ and heating for 3 rain at 100°C, the elec~rophoresis was run according to Shapiro et al. (1967), except for the staining, which was done by aniline black instead of coomassie blue. (D) Here the procedure of Carraway & Kobylka (1970) was followed. The membranes were dissolved in SDS-phosphate buffer (pH 7.8) containing mercaptoethanol and glycerol; electrophoresiswas run as indicated in ( C ) . . For the electrophoretic study of glycoproteins, membranes were solubilized according to Lenard (1970) and staining was d o ~ with periodic acid--Sch~. The molecular weights of proteins, sepmated to procedures 03) and (C), wen~.estimated with the u~e of trypsin (24,000), glyadin (27j)00), o ~ (44,000), seralbumin (65,000) and ~gl0bulins (160,000) as standard proteins. In some cases, the lipids of the erythrocyte raembranes were removed by a chloroform-methanol (2 : 1, v/v) extraction procedure, for 4 hr, several times. Hydrolysis for sialic acid was performed with 250 mg of erythroeyte ghosts (dry weight) in 10ml of 0-1N HISO, at 80°C, for 1 hr; the hydrolysis was repeated three or four times. Ba(OH)t was added Until the pH was 5.1, and the przcipitate was eliminated by centrifusation. After purification (Cabezas et al., 1964), sialic acids were identified by descending paper (Schieicher & Schfill
2043b) chromatography, with the use of the following solvents: (i) n-butanol-acetic acid-water ( 4 : 1 : 5 ) (72 hr); (ii) n-propanol-0.1N HCI ( 1 : 2 : 1) ( 4 8rh) ; (iii) n-~tanol-pyridine-0.1 N HC1 (5 : 3 : 2) (96 hr) (v/v). Sialic acids were located on paper with p-dimethylaminobenzaldehide (modified Ehrllch reagent) (Bezkorovalny, 1963). For the estimation of the ratio of each sialic acid, the respective zone of paper was eluted with a mixture of methanol-acetic acid (2 : 1) and the eluates read to 483 rim. In some cases, the Eegriwe test (Falllard & Cabezas, 1963) was used to determine N-glycolylneuraminic acid. The hydrolysis for hexoses was carried out with 1 N HC1 at 100°C for 4 hr; and for hexosamines, with 3 N HCI at 100°C for 4 hr. Oses and osamines were located on paper by a AgNes solution. For the quantitative determination of sailic acids, the modified resorcinol procedure (Cabezas et al., 1964) was applied Oses (with the use of galactosc-manncee, 1 : 1, as standard), bexosamines (with a standard of glucosamine chlorhydrate) and proteins were determined as previously reported (Cabezas et al., 1964, 1972). RESULTS AND DISCUSSION The best resolution in the proteins' electrophoretic patterns, obtained with the use of gel acrylamide, has been achieved with procedure (C) (Fig. 1). When procedures (A), (B) or (D) were employed the results were of less resolution and were difficult to interpret. The molecular weights for erythrocyte membrane proteins were estimated as ranging between 250,000 and 22,000. Figure 2 shows the pattern of glycoproteins, obtained essentially by procedure (C). As expected, the highest similarity for proteins and glycoproteins was observed between the horse, mule and donkey s a m p l e s ; the patterns among other species show more marked differences. A parallel result was previously found for serum proteins and glycoproteins of all the above-mentioned species (Cabezas et al., 1964, 1972). Some variations in the results reported by different authors could arise from variations in the preparation of the erythrocyte membranes; also, by the use o f very different agents for solubilization of the proteins and glycoproteins; and, finally, by the utilization o f several modalities of gel electrophoresis. On the other hand, it is known that the charge o f sialic acid play an important role in the mobility of the glycoproteins, and that this acid is located at the outer surface o f the cell. We have liberated all the siatic acid f r o m tbe erythrocyte membranes after two or three very mild hydrolyses; the concentration o f the released sialic acid decreases from the first to the second and third hydrolyses. The type and content of the sialic acids found in the above-mentioned species can be seen in Table 1. The same type of sialic acid, nearly 100~o N-glycolylneuraminic acid, has been detected in the horse, mule and donkey; in the cow and sheep, about a 80% o f sialic acids are N-glycolylneuraminic acid, with the remainder being N-acetylneuraminic acid; finally, in the pig there exist these acids and one other acid, probably a
Fig: 1. Acrylamide gel electrophoretic patterns of erythrocyte membrane proteins. The membranes were solubilized and the electrophoresis was carded out as explained in the text (procedure C). I, Horse; 2, mule; 3, donkey; 4, cow; 5, pig; 6, sheep. Fig. 2. Acrylamide gel electrophoretic patterns of erythrocyte membrane glyeoproteins. The membranes were solubilized and the electrophoresis was carried out as explained in the text. 1, Horse; 2, mule; 3, donkey; 4, cow; 5, pig; 6, sheep.