/. Biochem., 77, 919-930 (1975)
Sialoglycopeptides Isolated from Bovine. Aorta 1
Department of Biochemistry, Tohoku University School of Medicine, Sendai, Miyagi 980 Received for publication, November 22, 1974
Intima-media of bovine aorta was digested with pronase, after preliminary extraction of saline (l%)-soluble substances and fat. Crude glycopeptide fraction was then obtained from the resulting complex carbohydrate fraction, by fractionation with CPC (cetylpyridinium chloride). Complete separation of sialoglycopeptides was achieved by chromatography on a DEAE-cellulose column at pH 7.2 followed by repeated chromatography on a DEAE-Sephadex A-25 column at pH 5.2. Nine sialoglycopeptides (SGP 1-SGP 9) thus obtained were homogeneous on high-voltage paper electrophoresis at pH 3.5 and pH 5.2. The analytical data showed great heterogeneity of the carbohydrate chains of these preparations, although they consisted of the same monosaccharides (galactose, glucose, mannose, glucosamine, galactosamine, fucose, and sialic acid), except that SGP 1 lacked galactosamine. Heterogeneity was also observed" in-their peptide chains. It was noticed, however, that the contents of hexose, hexosamine, and aspartic acid of the fractions (SGP 3, SGP 4, and SGP 5) which eluted from the DEAE-Sephadex A-25 column at lower molarity of the eluting salt were higher than those of the fractions (SGP 7, SGP 8, and SGP 9) which eluted at higher molarity, while the contents of sialic acid and hydroxyamino acids were in an opposite relationship. Representative fractions (SGP 7 and SGP 9) of the latter contained many more alkali-sensitive linkages than those (SGP 3 and SGP 5) of the former, indicating the presence of many more 0glycosidic linkages between hydroxyamino acid(s) and sugar(s) in the latter than in the former. The sialoglycopeptides contained significant amounts of sialic acid, ranging from 10% (SGP 1) to 32.4% (SGP 8). The highest contents were in SGP 8 and SGP 9, which contained equimolar amounts of sialic acid and hexosamine. Furthermore, infrared spectra indicated the presence of sulfate groups in most of the sialoglycopeptides.
The presence of glycoproteins containing sialic acid (sialoglycoproteins) in the mammalian arterial wall has been demonstrated histologically ( / ) and chemically (2—21) by a number . 1 This study was supported in part by a grant from the Ministry of Education of Japan. Vol. 77, No. 5, 1975
919
of investigators. Some of these glycoproteins can readily be extracted with water (3) and with salt or buffer solutions (4—7, 15-17, 20), while others require more drastic procedures such as the use of urea, alkali, or proteases for solubilization (9-14, 18, 19, 21). The latter group is presumably more important in
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/919/2185450 by Duke University user on 11 January 2019
Hiroshi SAITO and Zensaku YOSIZAWA
920
H. SAITO and Z. YOSIZAWA Bovine aorta (intima-media) Extracted with 1% NaCl
I
I
Extract
Residue Extracted with ethanol, ethanol-ether (1 : 1), ether, in succession
I
I
Extract
Defatted residue Digested with pronase Pronase digest + Trichloroacetic acid (final cone. 8%)
I
Aorta—Fresh bovine aortae (Aorta thoracia and abdominalis) were obtained from a slaughterhouse and were stored at —20° until use. CPC (cetylpyridinium chloride) was purchased from K & K Laboratories, Plainview, New York. Pronase P was from Kaken Kagaku Kogyo Co., Ltd., Tokyo; DEAE-Cellulose from Serva Co.; DEAE-Sephadex A-25 and CMSephadex C-25 from Pharmacia Fine Chemicals. Three per cent SE-30 on Chromosorb W (AWDMCS) for gas chromatography was obtained from GAS-CHRO Kogyo Co., Ltd., Tokyo. Other materials were obtained from commercial sources. Separation of the Crude Glycopeptide Fraction from Bovine Aorta—An outline of the separation procedure is given in Scheme 1. Elimination of Saline-soluble Substances— Bovine aortae, stored at —20°, were thawed and freed from adventitia. Intima-media (780 g) was dissected into small pieces, and then homogenized in a Waring blender with 2 volumes of 1% NaCl at 4° for 3 min. The mixture was subsequently centrifuged at 1,000 xg for 10 min. Four extractions by the same procedure were carried out. The residues were finally extracted overnight with 10 volumes of fresh 1% NaCl under mechanical stirring at 4°. The residue thus obtained was washed several times with water to remove NaCl, and then stirred mechanically with 4 volumes of ethanol for 4 hr. After filtration through gauze, the tissues were further defatted by extraction with 4 volumes each of ethanol-
I
Supernatant
MATERIALS AND METHODS
Precipitate
Dialyzed +Ethanol (final cone 80%) & NaCl
I
Supernatant
I
Precipitate Dissolved in water Dialyzed, Lyophilized
Complex carbohydrate fraction Dissolved in 0.03 M NaCl + Cetylpyridinium chloride
I
I
Supernatant Precipitate +Ethanol (final cone. 80%) & NaCl
I
Supernatant
I
Precipitate Dissolved in water Dialyzed, Lyophilized Crude glycopeptide fraction
Scheme 1. An outline of the separation procedure for crude glycopeptide fraction from intima-media of bovine aorta.
ether ( 1 : 1 , by volume) and ether, in succession, with mechanical stirring for 4 hr. The final residues were dried in vacuo over paraffin. Pronase Digestion—The defatted dry powder (137 g) was suspended in 500 ml of 0.05 M Tris-HCl buffer (pH 8.0) containing 0.02 M CaCU, and incubated at 37° with 1.6 g of pronase P in the presence of a small amount of toluene for 20 hr. The pH of the incubation mixture was maintained at 8.0 with 1 M NaOH during the proteolysis. The incubation was continued for a further 20 hr, and 0.8 g of the enzyme was added to the incubation mixture / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/919/2185450 by Duke University user on 11 January 2019
preserving the integrity of the tissue than the former, and seems to play an important role in morphological changes of the tissue, such as the alterations in atherosclerosis or aging (9, 10, 21). Although some of these sialoglycoproteins have been isolated and characterized (9, 10, 14, 18, 19), more comprehensive studies of these substances are necessary to determine their significance. This paper reports the isolation, purification, and characterization of nine sialoglycopeptides from intima-media of bovine aorta after digestion with pronase.
SIALOGLYCOPEPTIDES FROM BOVINE AORTA
921
trifuged at 1,000 xg for 10 min, and the residue was redigested for 24 hr with 0.2 g of pronase as described above. The supernatants obtained by centrifugation were combined, and the pH was adjusted to 7.0, then the solution was concentrated to approximately 200 ml. Next, 50% aqueous trichloroacetic acid was added to the concentrate at 0° to give a final concentration of 8%, and the mixture was kept at 0° for 5 min. The precipitates were removed by centrifugation at 3,000Xg for 10 min, and the clear supernatant was dialyzed
Crude glycopeptide fraction Fractionated on a DEAE-cellulose column, eluting with Tris-HCl buffer (0.05 M, pH 7.2) and a linear gradient of 0-1 M KC1 in the same buffer, in succession
I
I
I
D2 D3 Dialyzed, Lyophilized Lyophilized D2 Treated with CM-Sephadex C-25 column in sodium acetate buffer (0.5 M, pH 5.2) Non-retarded fraction Dialyzed, Lyophilized Fr. D2-C Fractionated on a DEAE-Sephadex A-25 column, eluting with 0.05 M sodium acetate buffer (pH 5.2) and a linear gradient of 0-0.5 M KC1 in the same buffer, in succession Fractions 1-8
Dl
Combined fractions (1, 3, 5, and 7)
Combined fractions (-1 and 5)
r
I Fraction 5
Combined fractions (2, 4, 6, and 8)
Combined fractions (3 and 7)
r
I Fraction 7
Combined fractions (4 and 8)
Combined fractions (2, 6, and 8')
r
I
I
I
Fraction 6
Fraction 8'
Fraction 4 **
I
Fraction Fraction Fraction Fraction 8 I 1 2 3 * * I •• I** SGP8 SGP6 SGP9 SGP4 SGP5 SGP7 SGP1 • Rechromatographed SGP3on a DEAE-SephadexSGP2 A-25 column by the same procedure as above. ** Passed through a CM-Sephadex C-25 (H+ form) column in water, and the resulting nonretarded fraction was lyophilized.
1"
I"
I"
1"
I"
1
I'
Scheme 2. An outline of the fractionation procedure for sialoglycopeptides from crude glycopeptide fraction. Vol. 77, No. 5, 1975
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/919/2185450 by Duke University user on 11 January 2019
every 10 hr. Subsequently, the mixture was dialyzed in Visking tubing against running tap water for 6 hr, and then against 5 liters of distilled water for 4 hr. To the non-dialyzable fraction, 1 M Tris-HCl buffer (pH 8.0) and 1 M CaCl2 were added to give final concentrations of 0.05 and 0.02 M, respectively. Subsequently, pronase digestion was repeated by similar procedures using 0.8 g of the enzyme for 12 hr. The incubation was continued for a further 24 hr with the addition of 0.4 g of the enzyme every 12 hr. The mixture was cen-
922
H. SAITO and Z. YOSIZAWA
1—D1
D2ft
\
A
0.5
1
l>—03-H
f\ \
1 0 •
:
5 liters of distilled water for 12 hr. The nondialyzable fraction was then lyophilized, yielding a crude glycopeptide fraction. Fractionation of Sialogiycopeptidesfrom the Crude Glycopeptide Fraction—The fractionation procedures are outlined in Scheme 2. - Chromatography on DEAE-cellulose — A batch (181.5 mg) of the crude glycopeptide fraction was dissolved in 10 ml of 0.05 M TrisHC1 buffer (pH 7.2), and the solution was applied to a column (2.2x35 cm) of DEAE-cellulose previously equilibrated with the same buffer. The column was then washed with 240 ml of the same buffer. Linear gradient elution was carried out with 700 ml of 0—1M KC1 in the same buffer. Fractions of 8 ml were collected. The contents of hexose, sialic acid, and uronic acid in each fraction were determined and the absorbance at 235 nm was also recorded. Fractions of the same peak were pooled. Thus, three pooled fractions designated as Dl, D2, and D3 were obtained, as shown in Fig. 1. Of these fractions, the sialic acid-containing fraction (D2) was dialyzed against several changes of 5 liters each of distilled water, and the non-dialyzable fraction was lyophilized.
k
30
w
l\ 1
60 Tube No.
.- 1 0
90
120
Fig. 1. DEAE-Cellulose column chromatography of the crude glycopeptide fraction of intima-media of bovine aorta. Chromatography was carried out on a column (2.2x35cm) of DEAE-cellulose, eluting successively with 240ml of 0.05M Tris-HCl buffer (pH 7.2) and 700 ml of a linear gradient of 0-1 M KC1 in the same buffer. Fractions of 8 ml were collected. The contents of hexose ( ), sialic acid (O), uronic acid (A), and the absorbance at 235nm ( ) of each fraction were determined as described in the text. , KC1 concentration. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/919/2185450 by Duke University user on 11 January 2019
against running tap water for 24 hr, and then against several changes of 5 liters each of distilled, water - for 48 hr. The non-dialyzable fraction was concentrated to 100 ml. Absolute ethanol was added to the concentrate to make a final concentration of 80% in the presence of a small amount of NaCl. The precipitates thus formed were collected by centrifugation, and dissolved in 50 ml of water. The solution was dialyzed against 5 liters of distilled water for 12 hr. The non-dialyzable fraction was then lyophilized, yielding a complex carbohydrate fraction. Fractionation with CPC {cetylpyridinium chloride) —The above complex carbohydrate fraction (2.5 g) was dissolved in 200 ml of 0.03 M NaCl. Approximately 120 ml of 8% aqueous CPC in 0.03 M NaCl was added to the solution with stirring. The precipitates were removed by centrifugation, and the supernatant was concentrated to 20 ml. Ethanol was added to the concentrate to make a final concentration of 80% in the presence of a small amount of NaCl. The precipitates thus formed were collected by centrifugation, washed twice with 100 ml of ethanol, and then dissolved in 20 ml of water. The solution was dialyzed against
SIALOGLYCOPEPTIDES FROM BOVINE AORTA
Chromatography of Sialoglycopeptide Fractions on DEAE-Sephadex A-25—DEAE-Sephadex A-25 (Cl~ form) was washed exhaustively with 1 M sodium acetate, and then stirred with 1M sodium acetate buffer (pH 5.2), followed by equilibration with 0.05 M sodium acetate buffer (pH 5.2). The Sephadex was then packed into a column (2.2x50 cm), and the column was washed well with the same buffer. A portion (230 mg) of Fr. D2-C was dissolved in 10 ml of the same buffer, and applied to the column. The column was then washed with 300 ml of the same buffer. Linear gradient elution was carried out with 700 ml of 0—0.5 M KC1 in the same buffer. Fractions of 7 ml were collected. The contents of ninhydrin-reactive substances and sialic acid in each fraction were determined. The elution pattern is shown in Fig. 2 (Al). The resulting oddnumbered peak substances and even-numbered peak substances were pooled separately for rechromatography. The rechromatography was performed on a column (1.5x45 cm), eluting successively with 150 ml of 0.05 M sodium acetate buffer (pH 5.2) and 560 ml of a linear gradient of 0-0.5 M KC1 in the same buffer. The content of hexose and the absorbance at 235 nm of each fraction were determined. The elution patterns on rechromatography of the odd-numbered peak substances and the evennumbered peak substances are shown in A2-1 and A2-2, respectively, in Fig. 2. Further purification of each peak material was carried out by repeated rechromatography using the same procedures. The elution patterns thus
Vol. 77, No. 5, 1975
0.4 (Al)
05
2
OlZ
cO-3
10
30
50
70 Tube No
40
60 Tub* No.
90
110
130
100
Fig. 2. Chromatography of sialoglycopeptide fractions on a DEAE-Sephadex A-25 column. Chromatography was carried out on a column (2.2x50 cm for Al, and 1.5x45 cm for A2-1 and A2-2) of DEAE-Sephadex A-25, eluting successively with 0.05 M sodium acetate buffer (pH 5.2) and a linear gradient of 0-0.5 M KC1 in the same buffer, as described in the text The contents of ninhydrin-reactive substance (•••••••), sialic acid (—O—), hexose ( ) and the absorbance at 235 nm ( ) in each fraction were determined as described in the text. , KC1 concentration. Al, chromatography of Fr. D2-C; A2-1, rechromatography of the odd-numbered peak substances of A l ; A2-2, rechromatography of the even-numbered peak substances of Al.
obtained are shown in Fig. 3 (A3-1 and A3-2 for the odd-numbered peak substances and A3-3 and A3-4 for the even-numbered peak substances). Each peak substance was dialyzed against several changes of 2 liters each of distilled water, and the non-dialyzable fraction was lyophilized. Nine glycopeptide fractions were obtained. Treatment of Glycopeptide Fractions with CM-Sephadex—CM-Sephadex. C-25 (Na+ form)
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/919/2185450 by Duke University user on 11 January 2019
Treatment of D2 with CM-Sephadex—CMSephadex C-25 (H+ form) was equilibrated with 0.5 M sodium acetate buffer (pH 5.2) and packed into a column (2.6x45 cm). The column was washed exhaustively with the same buffer. A portion (300 mg) of lyophilized D2 was dissolved in 10 ml of the same buffer, and then applied to the column. The column was then washed with 500 ml of the same buffer. Fractions of 10 ml were collected. The non-retarded fractions showing an absorption at 235 nm were pooled, and then dialyzed against several changes of 5 liters each of distilled water. The non-dialyzable fraction was lyophilized, yielding Fr. D2-C.
923
924
H. SAITO and Z. YOSIZAWA
as
0.5
•
ijj
{ A 3-1 ) 0.3"
803 m
Sialoglycopeptides Isolated from Bovine. Aorta 1
Department of Biochemistry, Tohoku University School of Medicine, Sendai, Miyagi 980 Received for publication, November 22, 1974
Intima-media of bovine aorta was digested with pronase, after preliminary extraction of saline (l%)-soluble substances and fat. Crude glycopeptide fraction was then obtained from the resulting complex carbohydrate fraction, by fractionation with CPC (cetylpyridinium chloride). Complete separation of sialoglycopeptides was achieved by chromatography on a DEAE-cellulose column at pH 7.2 followed by repeated chromatography on a DEAE-Sephadex A-25 column at pH 5.2. Nine sialoglycopeptides (SGP 1-SGP 9) thus obtained were homogeneous on high-voltage paper electrophoresis at pH 3.5 and pH 5.2. The analytical data showed great heterogeneity of the carbohydrate chains of these preparations, although they consisted of the same monosaccharides (galactose, glucose, mannose, glucosamine, galactosamine, fucose, and sialic acid), except that SGP 1 lacked galactosamine. Heterogeneity was also observed" in-their peptide chains. It was noticed, however, that the contents of hexose, hexosamine, and aspartic acid of the fractions (SGP 3, SGP 4, and SGP 5) which eluted from the DEAE-Sephadex A-25 column at lower molarity of the eluting salt were higher than those of the fractions (SGP 7, SGP 8, and SGP 9) which eluted at higher molarity, while the contents of sialic acid and hydroxyamino acids were in an opposite relationship. Representative fractions (SGP 7 and SGP 9) of the latter contained many more alkali-sensitive linkages than those (SGP 3 and SGP 5) of the former, indicating the presence of many more 0glycosidic linkages between hydroxyamino acid(s) and sugar(s) in the latter than in the former. The sialoglycopeptides contained significant amounts of sialic acid, ranging from 10% (SGP 1) to 32.4% (SGP 8). The highest contents were in SGP 8 and SGP 9, which contained equimolar amounts of sialic acid and hexosamine. Furthermore, infrared spectra indicated the presence of sulfate groups in most of the sialoglycopeptides.
The presence of glycoproteins containing sialic acid (sialoglycoproteins) in the mammalian arterial wall has been demonstrated histologically ( / ) and chemically (2—21) by a number . 1 This study was supported in part by a grant from the Ministry of Education of Japan. Vol. 77, No. 5, 1975
919
of investigators. Some of these glycoproteins can readily be extracted with water (3) and with salt or buffer solutions (4—7, 15-17, 20), while others require more drastic procedures such as the use of urea, alkali, or proteases for solubilization (9-14, 18, 19, 21). The latter group is presumably more important in
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/919/2185450 by Duke University user on 11 January 2019
Hiroshi SAITO and Zensaku YOSIZAWA
920
H. SAITO and Z. YOSIZAWA Bovine aorta (intima-media) Extracted with 1% NaCl
I
I
Extract
Residue Extracted with ethanol, ethanol-ether (1 : 1), ether, in succession
I
I
Extract
Defatted residue Digested with pronase Pronase digest + Trichloroacetic acid (final cone. 8%)
I
Aorta—Fresh bovine aortae (Aorta thoracia and abdominalis) were obtained from a slaughterhouse and were stored at —20° until use. CPC (cetylpyridinium chloride) was purchased from K & K Laboratories, Plainview, New York. Pronase P was from Kaken Kagaku Kogyo Co., Ltd., Tokyo; DEAE-Cellulose from Serva Co.; DEAE-Sephadex A-25 and CMSephadex C-25 from Pharmacia Fine Chemicals. Three per cent SE-30 on Chromosorb W (AWDMCS) for gas chromatography was obtained from GAS-CHRO Kogyo Co., Ltd., Tokyo. Other materials were obtained from commercial sources. Separation of the Crude Glycopeptide Fraction from Bovine Aorta—An outline of the separation procedure is given in Scheme 1. Elimination of Saline-soluble Substances— Bovine aortae, stored at —20°, were thawed and freed from adventitia. Intima-media (780 g) was dissected into small pieces, and then homogenized in a Waring blender with 2 volumes of 1% NaCl at 4° for 3 min. The mixture was subsequently centrifuged at 1,000 xg for 10 min. Four extractions by the same procedure were carried out. The residues were finally extracted overnight with 10 volumes of fresh 1% NaCl under mechanical stirring at 4°. The residue thus obtained was washed several times with water to remove NaCl, and then stirred mechanically with 4 volumes of ethanol for 4 hr. After filtration through gauze, the tissues were further defatted by extraction with 4 volumes each of ethanol-
I
Supernatant
MATERIALS AND METHODS
Precipitate
Dialyzed +Ethanol (final cone 80%) & NaCl
I
Supernatant
I
Precipitate Dissolved in water Dialyzed, Lyophilized
Complex carbohydrate fraction Dissolved in 0.03 M NaCl + Cetylpyridinium chloride
I
I
Supernatant Precipitate +Ethanol (final cone. 80%) & NaCl
I
Supernatant
I
Precipitate Dissolved in water Dialyzed, Lyophilized Crude glycopeptide fraction
Scheme 1. An outline of the separation procedure for crude glycopeptide fraction from intima-media of bovine aorta.
ether ( 1 : 1 , by volume) and ether, in succession, with mechanical stirring for 4 hr. The final residues were dried in vacuo over paraffin. Pronase Digestion—The defatted dry powder (137 g) was suspended in 500 ml of 0.05 M Tris-HCl buffer (pH 8.0) containing 0.02 M CaCU, and incubated at 37° with 1.6 g of pronase P in the presence of a small amount of toluene for 20 hr. The pH of the incubation mixture was maintained at 8.0 with 1 M NaOH during the proteolysis. The incubation was continued for a further 20 hr, and 0.8 g of the enzyme was added to the incubation mixture / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/919/2185450 by Duke University user on 11 January 2019
preserving the integrity of the tissue than the former, and seems to play an important role in morphological changes of the tissue, such as the alterations in atherosclerosis or aging (9, 10, 21). Although some of these sialoglycoproteins have been isolated and characterized (9, 10, 14, 18, 19), more comprehensive studies of these substances are necessary to determine their significance. This paper reports the isolation, purification, and characterization of nine sialoglycopeptides from intima-media of bovine aorta after digestion with pronase.
SIALOGLYCOPEPTIDES FROM BOVINE AORTA
921
trifuged at 1,000 xg for 10 min, and the residue was redigested for 24 hr with 0.2 g of pronase as described above. The supernatants obtained by centrifugation were combined, and the pH was adjusted to 7.0, then the solution was concentrated to approximately 200 ml. Next, 50% aqueous trichloroacetic acid was added to the concentrate at 0° to give a final concentration of 8%, and the mixture was kept at 0° for 5 min. The precipitates were removed by centrifugation at 3,000Xg for 10 min, and the clear supernatant was dialyzed
Crude glycopeptide fraction Fractionated on a DEAE-cellulose column, eluting with Tris-HCl buffer (0.05 M, pH 7.2) and a linear gradient of 0-1 M KC1 in the same buffer, in succession
I
I
I
D2 D3 Dialyzed, Lyophilized Lyophilized D2 Treated with CM-Sephadex C-25 column in sodium acetate buffer (0.5 M, pH 5.2) Non-retarded fraction Dialyzed, Lyophilized Fr. D2-C Fractionated on a DEAE-Sephadex A-25 column, eluting with 0.05 M sodium acetate buffer (pH 5.2) and a linear gradient of 0-0.5 M KC1 in the same buffer, in succession Fractions 1-8
Dl
Combined fractions (1, 3, 5, and 7)
Combined fractions (-1 and 5)
r
I Fraction 5
Combined fractions (2, 4, 6, and 8)
Combined fractions (3 and 7)
r
I Fraction 7
Combined fractions (4 and 8)
Combined fractions (2, 6, and 8')
r
I
I
I
Fraction 6
Fraction 8'
Fraction 4 **
I
Fraction Fraction Fraction Fraction 8 I 1 2 3 * * I •• I** SGP8 SGP6 SGP9 SGP4 SGP5 SGP7 SGP1 • Rechromatographed SGP3on a DEAE-SephadexSGP2 A-25 column by the same procedure as above. ** Passed through a CM-Sephadex C-25 (H+ form) column in water, and the resulting nonretarded fraction was lyophilized.
1"
I"
I"
1"
I"
1
I'
Scheme 2. An outline of the fractionation procedure for sialoglycopeptides from crude glycopeptide fraction. Vol. 77, No. 5, 1975
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/919/2185450 by Duke University user on 11 January 2019
every 10 hr. Subsequently, the mixture was dialyzed in Visking tubing against running tap water for 6 hr, and then against 5 liters of distilled water for 4 hr. To the non-dialyzable fraction, 1 M Tris-HCl buffer (pH 8.0) and 1 M CaCl2 were added to give final concentrations of 0.05 and 0.02 M, respectively. Subsequently, pronase digestion was repeated by similar procedures using 0.8 g of the enzyme for 12 hr. The incubation was continued for a further 24 hr with the addition of 0.4 g of the enzyme every 12 hr. The mixture was cen-
922
H. SAITO and Z. YOSIZAWA
1—D1
D2ft
\
A
0.5
1
l>—03-H
f\ \
1 0 •
:
5 liters of distilled water for 12 hr. The nondialyzable fraction was then lyophilized, yielding a crude glycopeptide fraction. Fractionation of Sialogiycopeptidesfrom the Crude Glycopeptide Fraction—The fractionation procedures are outlined in Scheme 2. - Chromatography on DEAE-cellulose — A batch (181.5 mg) of the crude glycopeptide fraction was dissolved in 10 ml of 0.05 M TrisHC1 buffer (pH 7.2), and the solution was applied to a column (2.2x35 cm) of DEAE-cellulose previously equilibrated with the same buffer. The column was then washed with 240 ml of the same buffer. Linear gradient elution was carried out with 700 ml of 0—1M KC1 in the same buffer. Fractions of 8 ml were collected. The contents of hexose, sialic acid, and uronic acid in each fraction were determined and the absorbance at 235 nm was also recorded. Fractions of the same peak were pooled. Thus, three pooled fractions designated as Dl, D2, and D3 were obtained, as shown in Fig. 1. Of these fractions, the sialic acid-containing fraction (D2) was dialyzed against several changes of 5 liters each of distilled water, and the non-dialyzable fraction was lyophilized.
k
30
w
l\ 1
60 Tube No.
.- 1 0
90
120
Fig. 1. DEAE-Cellulose column chromatography of the crude glycopeptide fraction of intima-media of bovine aorta. Chromatography was carried out on a column (2.2x35cm) of DEAE-cellulose, eluting successively with 240ml of 0.05M Tris-HCl buffer (pH 7.2) and 700 ml of a linear gradient of 0-1 M KC1 in the same buffer. Fractions of 8 ml were collected. The contents of hexose ( ), sialic acid (O), uronic acid (A), and the absorbance at 235nm ( ) of each fraction were determined as described in the text. , KC1 concentration. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/919/2185450 by Duke University user on 11 January 2019
against running tap water for 24 hr, and then against several changes of 5 liters each of distilled, water - for 48 hr. The non-dialyzable fraction was concentrated to 100 ml. Absolute ethanol was added to the concentrate to make a final concentration of 80% in the presence of a small amount of NaCl. The precipitates thus formed were collected by centrifugation, and dissolved in 50 ml of water. The solution was dialyzed against 5 liters of distilled water for 12 hr. The non-dialyzable fraction was then lyophilized, yielding a complex carbohydrate fraction. Fractionation with CPC {cetylpyridinium chloride) —The above complex carbohydrate fraction (2.5 g) was dissolved in 200 ml of 0.03 M NaCl. Approximately 120 ml of 8% aqueous CPC in 0.03 M NaCl was added to the solution with stirring. The precipitates were removed by centrifugation, and the supernatant was concentrated to 20 ml. Ethanol was added to the concentrate to make a final concentration of 80% in the presence of a small amount of NaCl. The precipitates thus formed were collected by centrifugation, washed twice with 100 ml of ethanol, and then dissolved in 20 ml of water. The solution was dialyzed against
SIALOGLYCOPEPTIDES FROM BOVINE AORTA
Chromatography of Sialoglycopeptide Fractions on DEAE-Sephadex A-25—DEAE-Sephadex A-25 (Cl~ form) was washed exhaustively with 1 M sodium acetate, and then stirred with 1M sodium acetate buffer (pH 5.2), followed by equilibration with 0.05 M sodium acetate buffer (pH 5.2). The Sephadex was then packed into a column (2.2x50 cm), and the column was washed well with the same buffer. A portion (230 mg) of Fr. D2-C was dissolved in 10 ml of the same buffer, and applied to the column. The column was then washed with 300 ml of the same buffer. Linear gradient elution was carried out with 700 ml of 0—0.5 M KC1 in the same buffer. Fractions of 7 ml were collected. The contents of ninhydrin-reactive substances and sialic acid in each fraction were determined. The elution pattern is shown in Fig. 2 (Al). The resulting oddnumbered peak substances and even-numbered peak substances were pooled separately for rechromatography. The rechromatography was performed on a column (1.5x45 cm), eluting successively with 150 ml of 0.05 M sodium acetate buffer (pH 5.2) and 560 ml of a linear gradient of 0-0.5 M KC1 in the same buffer. The content of hexose and the absorbance at 235 nm of each fraction were determined. The elution patterns on rechromatography of the odd-numbered peak substances and the evennumbered peak substances are shown in A2-1 and A2-2, respectively, in Fig. 2. Further purification of each peak material was carried out by repeated rechromatography using the same procedures. The elution patterns thus
Vol. 77, No. 5, 1975
0.4 (Al)
05
2
OlZ
cO-3
10
30
50
70 Tube No
40
60 Tub* No.
90
110
130
100
Fig. 2. Chromatography of sialoglycopeptide fractions on a DEAE-Sephadex A-25 column. Chromatography was carried out on a column (2.2x50 cm for Al, and 1.5x45 cm for A2-1 and A2-2) of DEAE-Sephadex A-25, eluting successively with 0.05 M sodium acetate buffer (pH 5.2) and a linear gradient of 0-0.5 M KC1 in the same buffer, as described in the text The contents of ninhydrin-reactive substance (•••••••), sialic acid (—O—), hexose ( ) and the absorbance at 235 nm ( ) in each fraction were determined as described in the text. , KC1 concentration. Al, chromatography of Fr. D2-C; A2-1, rechromatography of the odd-numbered peak substances of A l ; A2-2, rechromatography of the even-numbered peak substances of Al.
obtained are shown in Fig. 3 (A3-1 and A3-2 for the odd-numbered peak substances and A3-3 and A3-4 for the even-numbered peak substances). Each peak substance was dialyzed against several changes of 2 liters each of distilled water, and the non-dialyzable fraction was lyophilized. Nine glycopeptide fractions were obtained. Treatment of Glycopeptide Fractions with CM-Sephadex—CM-Sephadex. C-25 (Na+ form)
Downloaded from https://academic.oup.com/jb/article-abstract/77/5/919/2185450 by Duke University user on 11 January 2019
Treatment of D2 with CM-Sephadex—CMSephadex C-25 (H+ form) was equilibrated with 0.5 M sodium acetate buffer (pH 5.2) and packed into a column (2.6x45 cm). The column was washed exhaustively with the same buffer. A portion (300 mg) of lyophilized D2 was dissolved in 10 ml of the same buffer, and then applied to the column. The column was then washed with 500 ml of the same buffer. Fractions of 10 ml were collected. The non-retarded fractions showing an absorption at 235 nm were pooled, and then dialyzed against several changes of 5 liters each of distilled water. The non-dialyzable fraction was lyophilized, yielding Fr. D2-C.
923
924
H. SAITO and Z. YOSIZAWA
as
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{ A 3-1 ) 0.3"
803 m
