Vol. 22. No. 6. pp. 653-658, 1990 Pnnted III Great Brilaln. All right? reserved
0020.711X:90
111,. J. Bmch~m.
Copyright
c: 1990
$3.00 + 0.00
Perpamon Press plc
ISOLATION AND PARTIAL CHARACTERIZATION OF TWO GLYCOPROTEINS FROM HUMAN LIVER METASTASES OF SIGMOID COLON CARCINOMA KATSUHIRO ‘Department
‘Pathology
Division.
OTSUKA,’
of Biochemistry,
National
SEIICHI OHKUMA’*
and
TAKASHI
Tokyo College of Pharmacy. Tokyo 192-03, Japan
Cancer
Research
Institute,
5-l-l
NAKAJIMA’
1432-I Horinouchi, Tsukiji,
Chuo-ku.
Hachioji,
Tokyo
104. Japan
(Received 9 Noaember 1989) Abstract-I.
Perchloric acid-soluble glycoprotein fraction (PASF) extracted from human liver metastases (LM) of sigmoid colon carcinoma was chromatographed on a DEAE-cellulose column. The main fraction (DEAE-nonadsorbed fraction) passed through the column was then subjected to Sephacryl S-200 superfine gel filtration and separated into 12 fractions. 2. Among 12 fractions, only both Fractions 3 and 4 were demonstrated to be chemically and immunologically homogeneous glycoproteins, respectively, by a combination of chemical composition analysis. SDS-PAGE and EITB assay using antisera against the DEAE-nonadsorbed fractions of PASFs from human LMs. normal liver (NL) and normal sigmoid colon (NSC). Each of Fractions 3 and 4 reacted with anti-LM serum to give one immuno complex on a nitrocellulose sheet in EITB assay, but did not react with anti-NL and -NSC sera. 3. Apparent molecular weights of 80,900 and 62,100, respectively, were found for Fractions 3 and 4. Both the fractions, respectively, had abnormal sugar compositions. Fraction 3 contained sialic acid. fucose. galactose, N-acetylglucosamine and N-acetylgalactosamine, but lacked glucose and mannose, and Fraction 4 contained sialic acid. fucose, galactose and N-acetylglucosamine. but lacked glucose, mannose and N-acetylgalactosamine. as sugar components.
INTRODUCTION
MATERIALS
LM from sigmoid colon carcinoma of a patient with blood group A. was obtained at autopsy within 10 hr until used. Reference of death and stored at -80’C proteins, chymotrypsinogen A (M,, 25,000), egg albumin (M,, 45,000) bovine serum albumin (M,, 68,000) and weight estimation catalase (M,, 240,000), in molecular were purchased from Boehringer Mannheim GmbH (Mannheim, West Germany). r-amylase was obtained from Sigma Chemical Co. (St Louis. MO., U.S.A.). DEAE~ellulose and Sephacryl S-200 superfine were obtained from Serva Feinbiochemica GmbH & Co. (Heidelberg, West Germany) and Pharmacia Fine Chemicals (Uppsala, Sweden), respectively. Complete Freund’s adjuvant and immun-blot protein A-HRP assay kit were purchased from Difco Laboratories (Detroit, Mich., U.S.A.) and Bio-Rad Laboratories (Richmond, Calif., U.S.A.), respectively.
Human liver carcinoma produce abnormally z-fetoprotein (Tatarinov, 1965), carcinoembryonic antigen (CEA) (Yamashita er al., 1987), novel Y-GTP (Sawabu et nl.. 1983) and variant alkaline phos-
phatase (Warnock and Reisman, 1969). These tumour-associated substances are known to be glycoproteins and commonly being used as important tumour markers in a clinical diagnosis of liver cancer (Urushizaki and Hattori, 1985; Cimino et al., 1987). On the other hand. a large number of tumourassociated glycoconjugates have been separated from human carcinomas such as gastric cancer, colon cancer, colorectal cancer. pancreatic cancer, hepatic adenocarcinoma. lung adenocarcinoma, melanoma and neuroectoderm, and the abnormal sugar chain structures of their glycoconjugates have been studied in many laboratories (Hakomori, 1984; Feizi, 1985; Feizi and Childs, 1987). Therefore, it is proper to suppose that abnormal glycoproteins other than the above-mentioned four tumour-associated substances occur in liver carcinomas. However, the presence or absence of such glycoproteins in liver carcinomas has not yet been examined. In this paper, we describe isolation and some chemical and immunological characterization of two glycoproteins with abnormal sugar compositions from human liver metastases (LM) of sigmoid colon carcinoma. *To whom
all correspondence
should
AND METHODS
Chemicul unulysis Sialic acid and neutral sugar were determined by the periodate-resorcinol reaction (Jourdian et al., 1971) and the phenol&sulphuric acid reaction (Dubois e/ crl., 1956), respectively. Hexosamines were determined by the modified Elson-Morgan reaction of Gatt and Berman (1966). Individual neutral sugars and hexosamines were determined by gas-liquid chromatography using a Shimadzu GC-YAM. after conversion of the component sugars to respective alditol acetates according to the method of Spiro (1972). Amino acids were determined by a Hitachi model L-8500 high-speed amino acid analyser after hydrolysis of a sample in 6 N HCI at 110 C for 22 hr. Protein was estimated either by the method (750 nm) of Lowry ef al. (1951) or by absorbance at 280 nm.
be addressed 653
KATSIJHIRO OTSUKA et al.
654
DEAE-nonadsorbed fraction
,
Fr.
II
,
,Fr. 111,
0.5 M NaC:
Fraction
Fig. 1. DEAEcellulose
column
chromatography
of PASF from human
SDS-PAGE SDSSPAGE was carried out according to the method of Sano (1979) in 15% polyacrylamide gel at pH 8.8. Bands separated were stained with the Coomassie Blue and PAS reagents (Zacharius et al., 1969) and their colour intensities were densitometrically measured by a Shimadzu CS-9000 DUAL-wavelength flying-spot scanner. Enqwe-induced
immunoelectrorransfer
blot (EITB)
assa?
After SDS-PAGE, glycoproteins were transferred from gels to nitrocellulose sheets by the method of Towbin et a/. (1979). Immuno-binding assay was performed according to the method of Hawkes et al. (1982) using anti-LM, -NL and -NSC sera and immunblot protein A-HRP assay kit. After EITB assay, the intensities of coloured bands of fractions 3 and 4 obtained from the main fraction (DEAE-nonadsorbed fraction) in Sephacryl S-200 gel filtration, were measured densitometrically by a Shimadzu CS-9000 DUAL-wavelength flying-spot scanner. Estimation qf molecular weight Apparent molecular weights of Fractions 3 and 4 were estimated by the method of Shapiro ef al. (1967) using SDSPAGE. Chymotrypsinogen A, egg albumin, bovine serum albumin and catalase were used as calibration proteins in molecular weight estimation. Preparation qf anti-LM,
No.
-NL and -NSC sera
The mixture of a solution of 1.0 mg of the main fraction (DEAE-nonadsorbed fraction) substance in DEAEcellulose column chromatography of perchloric acid-soluble glycoprotein fraction (PASF) in l.Oml of phosphate-buffered saline (pH 7.4, PBS) and 1 ml of complete Freund’s adjuvant was intramuscularly injected into the back and footpads of a White New Zealand rabbit weighing ca 2.5 kg. The three rabbits immunized were bled from their carotid artery at 4 weeks after the injection. The rabbit sera were mixed and permitted to stand at 56°C for 30 min. Anti-LM (titer 64), -NL (titer 64) and -NSC (titer 32) sera were obtained by immunizing rabbits with the DEAE-nonadsorbed fraction substances prepared from PASFs of human LM from sigmoid colon carcinoma, human normal liver (NL) and human normal sigmoid colon (NSC), respectively. The DEAE-nonadsorbed fraction substances prepared from PASFs of NL and NSC in a previous
study (Otsuka work.
LM of sigmoid
colon carctnoma
et al.. 1985) were used as immunogens
rn thus
Isolation of IWOglycoproteins fiorn human LM o/’ .\ry.mord colon carcinoma Human LM from sigmoid colon carcinoma was nnnced. homogenized in 4~01 of water in the presence of phenylmethylsulphonyl fluoride (Loor PI trl., 1981) and treated with perchloric acid in 1.0 M concentration according to the method of Krupey e/ ul. (1972). The perchloric acid extract obtained was neutralized. dialysed fully against disttlled water at 34 C and lyophilized. The dry residue was delipidized repeatedly with acetone, followed hy chloroform-methanol (2: I, v/v) mixture. The delipidized residue was treated three times with r-amylase by the method of Bernfeld (1955) to remove glycogen as a contaminant. The treated solution was deproteinized with perchloric acid, centrifuged, neutralized, dialysed fully against distilled water and lyophilized. The dry residue was named as PASF. A solution of 100 mg of PASF in 3.0 ml of 0.05 M sodium phosphate buffer (pH 7.5) was applied to a DEAE cellulose column (4.6 x 8.0 cm) previously equilibrated with the same buffer. Chromatography was carried out at 3 4 C. The column was washed with the same buffer and adsorbed matters were eluted with the same buffer containing 0.1 M NaCl and then with the same buffer containing 0.5 M NaCl at a flow rate of 30 ml/hr. as shown in Fig. 1, and 8 ml fractions were collected. Absorbances at 490 nm for neutral sugar and 750 nm for protein of each of the fractions were determined. The fractions showing absorbances at 750 and 490 nm were pooled, dialysed fully against distilled water at 34’C and lyophilized. The dry matter from the main fraction (Fraction I in Fig. 1) passed through the column was designated as DEAE-nonadsorbed fraction. A solution of 7.0 mg of DEAE-nonadsorbed fraction in 0.5 ml of 0.05 M sodium phosphate buffer (pH 7.5) containing 0.15 M NaCl and 0.02% NaN, was applied to a Sephacryl S-200 superfine column previously equilibrated with the same buffer. The column was washed and eluated with the same buffer at 34’C in a flow rate of 10 ml’hr. as shown in Fig. 2. and 5 ml fractions were collected. Absorbances at 490nm for neutral sugar and 750 nm for protein of each of the fractions were determined. The fractions showing absorbance at 490 nm were pooled. dialysed fully against distilled water at 3 4 C and lyophilized.
655
Liver metastases glycoproteins Table
I. Yields and chemical compositions of fractions separated from PASF by DEAE-cellulose column chromatography Fraction Fraction
Fraction
Fraction
PASF
I
II
111
199.0
89.9 45.2
43.0 21.6
33.0 16.6
1.71 3.44 3.84 85.83
I .54 2.77 3.65 90 66
3.45 5.91 4.18 78.78
I .38 5.30 7.25 56.78
Yield
mg. 100 g of wet tlbsue mg: 100 me of PASF CIIEI?lII.(II CunI,““iriiIn Slallc acid kutral sugar Hexowmme Protein
/%)
RESULTS
Estraction carcinoma
of‘PASF,fiom
human LM qf‘sigmoid colon
PASF was obtained from human LM of sigmoid colon carcinoma by using the perchloric acid extraction method of Krupey rt al. (1972) which was developed for the preparation of CEA from large quantities of human tumour tissues. Yield of PASF from human LM was 199 mg/lOO g of wet tissue (Table I). Chemical composition presented in Table 1 showed that PASF is a glycoprotein fraction. Separation g!,wproteins
and partial ,fiom PASF
characterization
of
two
PASF was subjected to DEAE-cellulose column chromatography using 0.05 M sodium phosphate buffer (pH 7.5) and the same buffers containing 0.1 M NaCl and 0.5 M NaCl as eluents, and separated into three fractions, Fractions I, II and III, as shown in Fig. 1. The three fractions were shown to be glycoproteins by their chemical composition analyses, as presented in Table I. Fraction I was evaluated as the main fraction by its chromatographic pattern (Fig. 1) and its yield (Table 1) to PASF. and designated as DEAE-nonadsorbed fraction. DEAE-nonadsorbed fraction was then gel
0.1
) 8
I
filtrated on a Sephacryl S-200 superfine column by using 0.05 M sodium phosphate buffer (pH 7.5) containing 0.15 M NaCl and 0.02% NaN,, and separated into I2 fractions, as presented in Fig. 2. Yields of these fraction are shown in Table 2. To detect the homogeneity of Fractions I-12, these fractions were analysed by SDS-PAGE using the Coomassie Brilliant Blue and PAS stainings and also by EITB assay with anti-LM, -NL and -NSC sera. Table 2 presents the analytical results of these fractions by SDS-PAGE and EITB assay. As shown in Table 2, among these fractions, Fractions I, 2 and 5-12 gave no single bands which react with both the Coomassie Brilliant Blue and PAS reagents on SDS gel plates and also with anti-LM serum on nitrocellulose sheets. In contrast to these 10 fractions, in SDS-PAGE, both Fractions 3 and 4 migrated anodically as single bands with different mobilities (R, values: Fraction 3, 0.16; Fraction 4, 0.21) and reacted with both the staining reagents, and on the one hand, both the fractions reacted with anti-LM serum to give single immuno complexes (R, values: Fraction 3, 0.16; Fraction 4, 0.21) on nitrocellulose sheets in EITB assay, respectively, but did not react with anti-NL and -NSC sera, as shown in Fig. 3. Apparent molecular weights of Fractions 3 and 4 were estimated by the method of Shapiro et al. (1967) using SDS-PAGE and shown to be 80,900 and 62,100, respectively, as presented in Fig. 4. As seen in Table 3. Fraction 3 showed a greater proportion of carbohydrate composition than Fraction 4. The contents of sialic acid and galactose of Fraction 3 were significantly higher than those of Fraction 4. Little difference in fucose content was found between Fractions 3 and 4. Neither of Fractions 3 and 4 contained glucose and mannose, and the latter fraction also lacked N-acetylgalactosamine as a sugar component. The content of aspartic acid, threonine and serine of Fraction 3 were significantly higher than those of Fraction 4.
Ft-. 6
2345
7
8
9
10
11
12
2
10
20
30
40
50 Fraction
60
70
80
No.
Fig. 2. Sephacryl S-200 superfine gel filtration of DEAE-nonadsorbed
fraction.
90
100
656
et al.
KATSUHIRO OTSUKA
Fr.
(GP. 1 3
Fr.
(0 16) Coomassie staining
SDS-PAGE
!
blue
PAS staining
(GP.)
4
(0 21)
I (0 16)
1
1
I
(0 16) Anti-LM ser~rn reaction EITB
Anti-NL serum reaction
Anti-NW serum reaction
1
0
05 Relative
Fig.
3. SDS -PAGE
I
i
1
0
10
mobility
05 Relative
10
mobility
and EITB assay patterns of Fractions (GPs) 3 and 4 separated nonadsorbed fraction by Sephacryl S-200 superfine gel filtration.
DlSCUSSlON
Krupey ef ul. (1972) stated that the perchloric acid extraction method is useful for the extraction of CEA, a sugar-rich glycoprotein, of the human digestive system from large quantities of metastatic tumour tissue. So, the method of Krupey et al. (1972) was applied to the extraction of glycoproteins from human LM of sigmoid colon carcinoma in this work and PASF was obtained in satisfactory yield from the metastatic tumour tissue. PASF was then subjected to DEAEcellulose column chromatography and separated into three fractions, Fractions I, II and III. as shown in Fig. 1. Table 1 presents yields and chemical compositions of the three fractions. The results show that these fractions are glycoproteins. respectively and that among the three fractions, Fraction I is the main fraction which was named as DEAE-nonadsorbed fraction, because the yield of this fraction was clearly higher than those of both Fractions II and III and this fraction passed through the DEAE
0020.711X:90
111,. J. Bmch~m.
Copyright
c: 1990
$3.00 + 0.00
Perpamon Press plc
ISOLATION AND PARTIAL CHARACTERIZATION OF TWO GLYCOPROTEINS FROM HUMAN LIVER METASTASES OF SIGMOID COLON CARCINOMA KATSUHIRO ‘Department
‘Pathology
Division.
OTSUKA,’
of Biochemistry,
National
SEIICHI OHKUMA’*
and
TAKASHI
Tokyo College of Pharmacy. Tokyo 192-03, Japan
Cancer
Research
Institute,
5-l-l
NAKAJIMA’
1432-I Horinouchi, Tsukiji,
Chuo-ku.
Hachioji,
Tokyo
104. Japan
(Received 9 Noaember 1989) Abstract-I.
Perchloric acid-soluble glycoprotein fraction (PASF) extracted from human liver metastases (LM) of sigmoid colon carcinoma was chromatographed on a DEAE-cellulose column. The main fraction (DEAE-nonadsorbed fraction) passed through the column was then subjected to Sephacryl S-200 superfine gel filtration and separated into 12 fractions. 2. Among 12 fractions, only both Fractions 3 and 4 were demonstrated to be chemically and immunologically homogeneous glycoproteins, respectively, by a combination of chemical composition analysis. SDS-PAGE and EITB assay using antisera against the DEAE-nonadsorbed fractions of PASFs from human LMs. normal liver (NL) and normal sigmoid colon (NSC). Each of Fractions 3 and 4 reacted with anti-LM serum to give one immuno complex on a nitrocellulose sheet in EITB assay, but did not react with anti-NL and -NSC sera. 3. Apparent molecular weights of 80,900 and 62,100, respectively, were found for Fractions 3 and 4. Both the fractions, respectively, had abnormal sugar compositions. Fraction 3 contained sialic acid. fucose. galactose, N-acetylglucosamine and N-acetylgalactosamine, but lacked glucose and mannose, and Fraction 4 contained sialic acid. fucose, galactose and N-acetylglucosamine. but lacked glucose, mannose and N-acetylgalactosamine. as sugar components.
INTRODUCTION
MATERIALS
LM from sigmoid colon carcinoma of a patient with blood group A. was obtained at autopsy within 10 hr until used. Reference of death and stored at -80’C proteins, chymotrypsinogen A (M,, 25,000), egg albumin (M,, 45,000) bovine serum albumin (M,, 68,000) and weight estimation catalase (M,, 240,000), in molecular were purchased from Boehringer Mannheim GmbH (Mannheim, West Germany). r-amylase was obtained from Sigma Chemical Co. (St Louis. MO., U.S.A.). DEAE~ellulose and Sephacryl S-200 superfine were obtained from Serva Feinbiochemica GmbH & Co. (Heidelberg, West Germany) and Pharmacia Fine Chemicals (Uppsala, Sweden), respectively. Complete Freund’s adjuvant and immun-blot protein A-HRP assay kit were purchased from Difco Laboratories (Detroit, Mich., U.S.A.) and Bio-Rad Laboratories (Richmond, Calif., U.S.A.), respectively.
Human liver carcinoma produce abnormally z-fetoprotein (Tatarinov, 1965), carcinoembryonic antigen (CEA) (Yamashita er al., 1987), novel Y-GTP (Sawabu et nl.. 1983) and variant alkaline phos-
phatase (Warnock and Reisman, 1969). These tumour-associated substances are known to be glycoproteins and commonly being used as important tumour markers in a clinical diagnosis of liver cancer (Urushizaki and Hattori, 1985; Cimino et al., 1987). On the other hand. a large number of tumourassociated glycoconjugates have been separated from human carcinomas such as gastric cancer, colon cancer, colorectal cancer. pancreatic cancer, hepatic adenocarcinoma. lung adenocarcinoma, melanoma and neuroectoderm, and the abnormal sugar chain structures of their glycoconjugates have been studied in many laboratories (Hakomori, 1984; Feizi, 1985; Feizi and Childs, 1987). Therefore, it is proper to suppose that abnormal glycoproteins other than the above-mentioned four tumour-associated substances occur in liver carcinomas. However, the presence or absence of such glycoproteins in liver carcinomas has not yet been examined. In this paper, we describe isolation and some chemical and immunological characterization of two glycoproteins with abnormal sugar compositions from human liver metastases (LM) of sigmoid colon carcinoma. *To whom
all correspondence
should
AND METHODS
Chemicul unulysis Sialic acid and neutral sugar were determined by the periodate-resorcinol reaction (Jourdian et al., 1971) and the phenol&sulphuric acid reaction (Dubois e/ crl., 1956), respectively. Hexosamines were determined by the modified Elson-Morgan reaction of Gatt and Berman (1966). Individual neutral sugars and hexosamines were determined by gas-liquid chromatography using a Shimadzu GC-YAM. after conversion of the component sugars to respective alditol acetates according to the method of Spiro (1972). Amino acids were determined by a Hitachi model L-8500 high-speed amino acid analyser after hydrolysis of a sample in 6 N HCI at 110 C for 22 hr. Protein was estimated either by the method (750 nm) of Lowry ef al. (1951) or by absorbance at 280 nm.
be addressed 653
KATSIJHIRO OTSUKA et al.
654
DEAE-nonadsorbed fraction
,
Fr.
II
,
,Fr. 111,
0.5 M NaC:
Fraction
Fig. 1. DEAEcellulose
column
chromatography
of PASF from human
SDS-PAGE SDSSPAGE was carried out according to the method of Sano (1979) in 15% polyacrylamide gel at pH 8.8. Bands separated were stained with the Coomassie Blue and PAS reagents (Zacharius et al., 1969) and their colour intensities were densitometrically measured by a Shimadzu CS-9000 DUAL-wavelength flying-spot scanner. Enqwe-induced
immunoelectrorransfer
blot (EITB)
assa?
After SDS-PAGE, glycoproteins were transferred from gels to nitrocellulose sheets by the method of Towbin et a/. (1979). Immuno-binding assay was performed according to the method of Hawkes et al. (1982) using anti-LM, -NL and -NSC sera and immunblot protein A-HRP assay kit. After EITB assay, the intensities of coloured bands of fractions 3 and 4 obtained from the main fraction (DEAE-nonadsorbed fraction) in Sephacryl S-200 gel filtration, were measured densitometrically by a Shimadzu CS-9000 DUAL-wavelength flying-spot scanner. Estimation qf molecular weight Apparent molecular weights of Fractions 3 and 4 were estimated by the method of Shapiro ef al. (1967) using SDSPAGE. Chymotrypsinogen A, egg albumin, bovine serum albumin and catalase were used as calibration proteins in molecular weight estimation. Preparation qf anti-LM,
No.
-NL and -NSC sera
The mixture of a solution of 1.0 mg of the main fraction (DEAE-nonadsorbed fraction) substance in DEAEcellulose column chromatography of perchloric acid-soluble glycoprotein fraction (PASF) in l.Oml of phosphate-buffered saline (pH 7.4, PBS) and 1 ml of complete Freund’s adjuvant was intramuscularly injected into the back and footpads of a White New Zealand rabbit weighing ca 2.5 kg. The three rabbits immunized were bled from their carotid artery at 4 weeks after the injection. The rabbit sera were mixed and permitted to stand at 56°C for 30 min. Anti-LM (titer 64), -NL (titer 64) and -NSC (titer 32) sera were obtained by immunizing rabbits with the DEAE-nonadsorbed fraction substances prepared from PASFs of human LM from sigmoid colon carcinoma, human normal liver (NL) and human normal sigmoid colon (NSC), respectively. The DEAE-nonadsorbed fraction substances prepared from PASFs of NL and NSC in a previous
study (Otsuka work.
LM of sigmoid
colon carctnoma
et al.. 1985) were used as immunogens
rn thus
Isolation of IWOglycoproteins fiorn human LM o/’ .\ry.mord colon carcinoma Human LM from sigmoid colon carcinoma was nnnced. homogenized in 4~01 of water in the presence of phenylmethylsulphonyl fluoride (Loor PI trl., 1981) and treated with perchloric acid in 1.0 M concentration according to the method of Krupey e/ ul. (1972). The perchloric acid extract obtained was neutralized. dialysed fully against disttlled water at 34 C and lyophilized. The dry residue was delipidized repeatedly with acetone, followed hy chloroform-methanol (2: I, v/v) mixture. The delipidized residue was treated three times with r-amylase by the method of Bernfeld (1955) to remove glycogen as a contaminant. The treated solution was deproteinized with perchloric acid, centrifuged, neutralized, dialysed fully against distilled water and lyophilized. The dry residue was named as PASF. A solution of 100 mg of PASF in 3.0 ml of 0.05 M sodium phosphate buffer (pH 7.5) was applied to a DEAE cellulose column (4.6 x 8.0 cm) previously equilibrated with the same buffer. Chromatography was carried out at 3 4 C. The column was washed with the same buffer and adsorbed matters were eluted with the same buffer containing 0.1 M NaCl and then with the same buffer containing 0.5 M NaCl at a flow rate of 30 ml/hr. as shown in Fig. 1, and 8 ml fractions were collected. Absorbances at 490 nm for neutral sugar and 750 nm for protein of each of the fractions were determined. The fractions showing absorbances at 750 and 490 nm were pooled, dialysed fully against distilled water at 34’C and lyophilized. The dry matter from the main fraction (Fraction I in Fig. 1) passed through the column was designated as DEAE-nonadsorbed fraction. A solution of 7.0 mg of DEAE-nonadsorbed fraction in 0.5 ml of 0.05 M sodium phosphate buffer (pH 7.5) containing 0.15 M NaCl and 0.02% NaN, was applied to a Sephacryl S-200 superfine column previously equilibrated with the same buffer. The column was washed and eluated with the same buffer at 34’C in a flow rate of 10 ml’hr. as shown in Fig. 2. and 5 ml fractions were collected. Absorbances at 490nm for neutral sugar and 750 nm for protein of each of the fractions were determined. The fractions showing absorbance at 490 nm were pooled. dialysed fully against distilled water at 3 4 C and lyophilized.
655
Liver metastases glycoproteins Table
I. Yields and chemical compositions of fractions separated from PASF by DEAE-cellulose column chromatography Fraction Fraction
Fraction
Fraction
PASF
I
II
111
199.0
89.9 45.2
43.0 21.6
33.0 16.6
1.71 3.44 3.84 85.83
I .54 2.77 3.65 90 66
3.45 5.91 4.18 78.78
I .38 5.30 7.25 56.78
Yield
mg. 100 g of wet tlbsue mg: 100 me of PASF CIIEI?lII.(II CunI,““iriiIn Slallc acid kutral sugar Hexowmme Protein
/%)
RESULTS
Estraction carcinoma
of‘PASF,fiom
human LM qf‘sigmoid colon
PASF was obtained from human LM of sigmoid colon carcinoma by using the perchloric acid extraction method of Krupey rt al. (1972) which was developed for the preparation of CEA from large quantities of human tumour tissues. Yield of PASF from human LM was 199 mg/lOO g of wet tissue (Table I). Chemical composition presented in Table 1 showed that PASF is a glycoprotein fraction. Separation g!,wproteins
and partial ,fiom PASF
characterization
of
two
PASF was subjected to DEAE-cellulose column chromatography using 0.05 M sodium phosphate buffer (pH 7.5) and the same buffers containing 0.1 M NaCl and 0.5 M NaCl as eluents, and separated into three fractions, Fractions I, II and III, as shown in Fig. 1. The three fractions were shown to be glycoproteins by their chemical composition analyses, as presented in Table I. Fraction I was evaluated as the main fraction by its chromatographic pattern (Fig. 1) and its yield (Table 1) to PASF. and designated as DEAE-nonadsorbed fraction. DEAE-nonadsorbed fraction was then gel
0.1
) 8
I
filtrated on a Sephacryl S-200 superfine column by using 0.05 M sodium phosphate buffer (pH 7.5) containing 0.15 M NaCl and 0.02% NaN,, and separated into I2 fractions, as presented in Fig. 2. Yields of these fraction are shown in Table 2. To detect the homogeneity of Fractions I-12, these fractions were analysed by SDS-PAGE using the Coomassie Brilliant Blue and PAS stainings and also by EITB assay with anti-LM, -NL and -NSC sera. Table 2 presents the analytical results of these fractions by SDS-PAGE and EITB assay. As shown in Table 2, among these fractions, Fractions I, 2 and 5-12 gave no single bands which react with both the Coomassie Brilliant Blue and PAS reagents on SDS gel plates and also with anti-LM serum on nitrocellulose sheets. In contrast to these 10 fractions, in SDS-PAGE, both Fractions 3 and 4 migrated anodically as single bands with different mobilities (R, values: Fraction 3, 0.16; Fraction 4, 0.21) and reacted with both the staining reagents, and on the one hand, both the fractions reacted with anti-LM serum to give single immuno complexes (R, values: Fraction 3, 0.16; Fraction 4, 0.21) on nitrocellulose sheets in EITB assay, respectively, but did not react with anti-NL and -NSC sera, as shown in Fig. 3. Apparent molecular weights of Fractions 3 and 4 were estimated by the method of Shapiro et al. (1967) using SDS-PAGE and shown to be 80,900 and 62,100, respectively, as presented in Fig. 4. As seen in Table 3. Fraction 3 showed a greater proportion of carbohydrate composition than Fraction 4. The contents of sialic acid and galactose of Fraction 3 were significantly higher than those of Fraction 4. Little difference in fucose content was found between Fractions 3 and 4. Neither of Fractions 3 and 4 contained glucose and mannose, and the latter fraction also lacked N-acetylgalactosamine as a sugar component. The content of aspartic acid, threonine and serine of Fraction 3 were significantly higher than those of Fraction 4.
Ft-. 6
2345
7
8
9
10
11
12
2
10
20
30
40
50 Fraction
60
70
80
No.
Fig. 2. Sephacryl S-200 superfine gel filtration of DEAE-nonadsorbed
fraction.
90
100
656
et al.
KATSUHIRO OTSUKA
Fr.
(GP. 1 3
Fr.
(0 16) Coomassie staining
SDS-PAGE
!
blue
PAS staining
(GP.)
4
(0 21)
I (0 16)
1
1
I
(0 16) Anti-LM ser~rn reaction EITB
Anti-NL serum reaction
Anti-NW serum reaction
1
0
05 Relative
Fig.
3. SDS -PAGE
I
i
1
0
10
mobility
05 Relative
10
mobility
and EITB assay patterns of Fractions (GPs) 3 and 4 separated nonadsorbed fraction by Sephacryl S-200 superfine gel filtration.
DlSCUSSlON
Krupey ef ul. (1972) stated that the perchloric acid extraction method is useful for the extraction of CEA, a sugar-rich glycoprotein, of the human digestive system from large quantities of metastatic tumour tissue. So, the method of Krupey et al. (1972) was applied to the extraction of glycoproteins from human LM of sigmoid colon carcinoma in this work and PASF was obtained in satisfactory yield from the metastatic tumour tissue. PASF was then subjected to DEAEcellulose column chromatography and separated into three fractions, Fractions I, II and III. as shown in Fig. 1. Table 1 presents yields and chemical compositions of the three fractions. The results show that these fractions are glycoproteins. respectively and that among the three fractions, Fraction I is the main fraction which was named as DEAE-nonadsorbed fraction, because the yield of this fraction was clearly higher than those of both Fractions II and III and this fraction passed through the DEAE
