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Comparative studies on the gastric glycopeptide in eleven animal species
Comp. Biochem. Physiol., 1977, Vol. 58B, pp. 163 to 165. Pergamon Press. Printed in Great Britain
COMPARATIVE STUDIES ON THE GASTRIC GLYCOPEPTIDE IN ELEVEN ANIMAL SPECIES H. MASUDA, S. SHICHIJO AND M. TAKEUCHI Institute of Clinical Pathology, Kurume University School of Medicine, Kurume, Fukuoka 830, Japan (Received 6 January 1977) Abstract--1. Glycopeptides in the stomachs of eleven mammalian species, including human, rabbit, horse, cow, pig, goat, sheep, dog, cat, guinea pig and rat were assayed by determining the carbohydrate content of materials which remained after proteolysis. 2. The glycopeptide content was higher in the mucosa than in the muscular layer including serosa, especially in the porcine stomach and the fourth stomachs of the ruminants than in the stomachs of any other animals. 3. The glycopeptide, which was stained with both alcian blue and PAS, was absent or sparingly present in the mucosae of the human, rabbit, horse stomachs and in the mucosae of the first to third stomachs of the cow, goat and sheep, whereas in the mucosae of the pig, dog, cat, guinea pig and rat stomachs and in the mucosae of the fourth stomachs of the cow, goat and sheep, it was found in noticeable extents.
INTRODUCTION It is believed that carbohydrates on cell surfaces may play important roles in many biological functions of the plasma membranes. The glycopeptides in the gastrointestinal tracts, including gastric mucosae of dog, pig and human have been analyzed, using chemical and histochemical techniques (Hakkinen et al., 1965; Hough & Jones, 1972; Slomiany & Meyer, 1972). At present, however, further work about these substances in the stomachs of other animals have not been reported. We have studied the variety of the content and composition of the glycopeptide in the stomachs of eleven male mammalian species. The stomachs were taken from 5 humans, rabbits, horses, cows, pigs, goats, sheep, dogs, cats, guinea pigs and rats. MATERIALS AND METHODS
The stomachs from humans were obtained at autopsy, and those of horses, cows, pigs, goats and sheep were from the local abbattoir, while those of rabbits, dogs, cats, guinea pigs and rats have been removed in the laboratory. The gastric wails were separated into two different layers; mucosa layer and muscular layer including serosa. The stomachs from cows, goats and sheep, however, were separated into bits of the first, second, third and fourth stomachs. They were cut into cherry-sized pieces, put into several changes of acetone for several days and dried in a CaCI2 desiccator. They were ground in a Wiley mill sufficiently small to pass through a 60 mesh sieve. All stomach tissues were treated similarly and compared one with another. Crude glycopeptide was extracted from dry tissue powder by the method of Schiller et al. (1961). It was digested with papain [EC 3.4.4.10] in the presence of EDTA and cysteine at 65°C for 48 hr. The mixture was adjusted to pH 7.8 and then digested with trypsin I-EC 3.4.4.4] at 37°C for 72 hr with simultaneous dialysis against 0.1 M phosphate buffer of pH 7.8. The digested solution was deproteinized with 5% trichloroacetic acid in the cold
and the supernatant was dialyzed against several changes of cold deionized water for 72 hr. Then 4 vol of alcohol containing 1% acetic acid and 1% potassium acetate were added to precipitate the crude glycopeptide and glycosaminoglycan. The precipitate was washed with alcohol and ether, dried in a CaCI2 desiccator and then redissolved in 20 ml of deionized water. An aliquot was used for hexose, sialic acid and uronic acid analyses. Hexose was determined by the orcinol-sulfuric acid reaction of Winzler (1955); sialic acid by the diphenylamine method of Winzler (1955); and uronic acid by the carbazole reaction of Dische (1962), respectively. Cellulose acetate electrophoresis of the material was carried out using barbital buffer, pH 8.6,/~ = 0.06. Strips were run in duplicates for 30 min with a current of 1 mA per cm width. After electrophoresis, strips were stained with both alcian blue and PAS. Marker samples containing hyaluronic acid, heparan sulfate, chondroitin sulfate-A, chondroitin sulfate-B and chondroitin sulfate-C were applied alongside each gastric material; hyaluronic acid, chondroitin sulfate-A, -B and -C were purchased from Sigma (U.S.A.), and heparan sulfate was kindly given by Dr A. Cifonelli (Chicago University, U.S.A.), respectively. The species used were: Human, Homo sapiens; rabbit, Oryctolaffus cuniculus; horse, Equus caballus, cow, Bos taurus; pig, Sus scrofa; goat, Capra hircus; sheep, Ovis aries; dog, Canis familiaris; cat, Felis catus; guinea pig, Cavia cobaya; rat, Rattus norve#icus. RESULTS
AND DISCUSSION
Results of the hexose, sialic acid and uronic acid analyses of the crude glycopeptide and glycosaminoglycans from the human, equine, bovine and porcine gastric walls are summarized in Table 1. Both the hexose and sialic acid contents of the mucosae were higher in the porcine stomach than in the human and equine stomach§. Concerning both the hexose and sialic acid contents of the mucosae from bovine stomachs, the values were highest in the fourth stomach among four kinds of stomachs, and similar
163
164
H . MASUDA, S. SHICHIJO AND M. TAKEUCHI TABLE 1. GLYCOPEPTIDE AND GLYCOSAM]NOGLYCAN CONTENTS IN THE STOMACH OF SEVERAL ANIMALS*
Species
Layer
H u m a n stomach
Mucosa Muscular-serosa Mucosa Museular-serosa Mucosa Museular-serosa Mucosa Muscular-serosa Mucosa Muscular-serosa Mucosa Muscular-serosa Mucosa Muscular-serosa
Equine stomach Bovine first stomach Bovine second stomach Bovine third stomach Bovine fourth stomach Porcine stomach
Hexose 6.3 2.8 5.3 3.3 4.3 1.4 3.3 1.7 5.5 2.9 13.0 2.3 17.9 3.0
-I- 1.62 ___ 1.00 _ 1.21 _ 1.24 _ 0.69 + 0.61 + 0.92 _ 0.52 ± 1.65 + 0.18 + 4.67 _ 1.21 _ 0.63 + 0.60
Sialic acid 0.5 0.4 0.5 0.5 0.5 0.4 0.4 0.4 0.7 0.6 1.2 0.4 1.3 0.4
+ 0.09 _ 0.10 _ 0.08 + 0.07 + 0.15 + 0.21 + 0.23 + 0.14 _ 0.34 + 0.02 ___ 0.22 + 0.23 + 0.14 + 0.09
Uronic acid 0.6 0.6 1.0 0.8 0.8 0.7 0.6 0.6 1.1 0.9 1.7 0.8 1.5 0.6
+ 0.07 + 0.09 ± 0.17 + 0.12 + 0.17 + 0.15 + 0.15 + 0.18 + 0.68 + 0.04 _ 0.17]" ___ 0.23 _ 0.12t + 0.09
* Expressed as mg/1 g of dry tissue. t Although the uronic acid contents of the swine and the bovine fourth stomach mucosae were higher than another stomach, the carbazole reaction of the materials from these two mucosae gave a brown colour which was not typical of uronic acid, and so might probably have more uronic acid than correct content.
PAS stain
Alcian blue stain
5 4 5 6 7 HA
Serum Ponceau-S)
HS hS I
0
Anode
Anode
Fig. 1. Electrophoretic patterns of the glycopeptide and glycosaminoglycans from the gastric mucosae on cellulose acetate strip. 1. H u m a n stomach; 2. Equine stomach; 3. Bovine first stomach; 4. Bovine second stomach; 5. Bovine third stomach; 6. Bovine fourth stomach; 7. Porcine stomach; HA. Hyaluronic acid; HS. Heparan sulfate; ChS. A mixture of chondroitin sulfate-A, -B and -C.
Comparative studies on the gastric glycopeptide results were obtained by the similar studies on the stomachs of both goat and sheep. Cellulose acetate strip-electrophoresis of materials from the mucosae revealed the presence of one PAS positive and three alcianophilic components in human, equine and bovine first to third stomachs. The former moved slightly to the anode, and the latter components migrated rapidly toward the anode and were identified as hyaluronic acid, heparan sulfate and chondroitin sulfates, as reported previously (Kimoto et al., 1965). In both bovine fourth and porcine stomachs, another component was also found which stained with both PAS and alcian blue. It migrated toward the anode slower than" hyaluronic acid (Fig. 1). Stomach tissues of rabbit, horse, dog, cat, guinea pig, rat, goat and sheep were also studied similarly. The glycopeptide, which was stained with both PAS and alcian blue, were absent or sparingly present in the mucosae of the rabbit, horse and first to third stomachs of the goat and sheep, whereas in the mucosae of the dog, cat, guinea pig, rat and the fourth stomachs of the goat and sheep, it was found to noticeable extents. And furthermore, it was not observed in the gastric muscular-serosa of any animals of the eleven species tested. Hakkinen et al. (1965) and Slomiany et al. (1972) isolated, after proteolytic digestion, sulfated glycopeptides from the dog and porcine gastric mucosae, respectively. Further investigation will be necessary to determine how the
165
glycopeptide from gastric mucosa of each animal differs biochemically from those from different kinds of animals.
REFERENCES
DISCHE Z. (1962) Color reactions of carbohydrates. In Methods in Carbohydrate Chemistry. (Edited by WHISTLERR. L. H WOLFORMM. L.), Vol, 1, pp. 497-501. Academic Press, New York. HAKKINENI., HATIALAK. H TERHOT. (1965) The fractionation and characterization of the acid polysaccharides of the gastric wall of the dog. Acta chem. scand. 19, 800-806. HOUGH L. & JONES J. V. S. (1972) Human gastric mucosa part 1. The preparation of a glycopolypeptide and some aspects of its structure. Carbohydrate Res. 23, 1-16. KIMOTOE., TANAKAY., KURANARIT., MASUDAH. & TAKEtJCm M. 0965) Sialomucopolysaccharides of human cancerous stomach. Proc. Syrup. Chem. Physiol. 5, 122-128. SCHILLER S., SLOVER G. A. & DORFMANN A. (1961) A method for the separation of acid mucopolysaecharides; Its application to the isolation of heparin from the skin of rats. J. biol. Chem. 236, 983-990. SLOMIANYB. L. H MEYER K. (1972) Isolation and structural studies of sulfated glycoproteins of hog gastric mucosa. J. biol. Chem. 247, 5062-5070. WINZLER R. J. (1955) Determination of serum glycoproteins. In Methods of Biochemical Analysis (Edited by GLICK D.), Vol. 2. pp. 279-324. Interscience, New York.
COMPARATIVE STUDIES ON THE GASTRIC GLYCOPEPTIDE IN ELEVEN ANIMAL SPECIES H. MASUDA, S. SHICHIJO AND M. TAKEUCHI Institute of Clinical Pathology, Kurume University School of Medicine, Kurume, Fukuoka 830, Japan (Received 6 January 1977) Abstract--1. Glycopeptides in the stomachs of eleven mammalian species, including human, rabbit, horse, cow, pig, goat, sheep, dog, cat, guinea pig and rat were assayed by determining the carbohydrate content of materials which remained after proteolysis. 2. The glycopeptide content was higher in the mucosa than in the muscular layer including serosa, especially in the porcine stomach and the fourth stomachs of the ruminants than in the stomachs of any other animals. 3. The glycopeptide, which was stained with both alcian blue and PAS, was absent or sparingly present in the mucosae of the human, rabbit, horse stomachs and in the mucosae of the first to third stomachs of the cow, goat and sheep, whereas in the mucosae of the pig, dog, cat, guinea pig and rat stomachs and in the mucosae of the fourth stomachs of the cow, goat and sheep, it was found in noticeable extents.
INTRODUCTION It is believed that carbohydrates on cell surfaces may play important roles in many biological functions of the plasma membranes. The glycopeptides in the gastrointestinal tracts, including gastric mucosae of dog, pig and human have been analyzed, using chemical and histochemical techniques (Hakkinen et al., 1965; Hough & Jones, 1972; Slomiany & Meyer, 1972). At present, however, further work about these substances in the stomachs of other animals have not been reported. We have studied the variety of the content and composition of the glycopeptide in the stomachs of eleven male mammalian species. The stomachs were taken from 5 humans, rabbits, horses, cows, pigs, goats, sheep, dogs, cats, guinea pigs and rats. MATERIALS AND METHODS
The stomachs from humans were obtained at autopsy, and those of horses, cows, pigs, goats and sheep were from the local abbattoir, while those of rabbits, dogs, cats, guinea pigs and rats have been removed in the laboratory. The gastric wails were separated into two different layers; mucosa layer and muscular layer including serosa. The stomachs from cows, goats and sheep, however, were separated into bits of the first, second, third and fourth stomachs. They were cut into cherry-sized pieces, put into several changes of acetone for several days and dried in a CaCI2 desiccator. They were ground in a Wiley mill sufficiently small to pass through a 60 mesh sieve. All stomach tissues were treated similarly and compared one with another. Crude glycopeptide was extracted from dry tissue powder by the method of Schiller et al. (1961). It was digested with papain [EC 3.4.4.10] in the presence of EDTA and cysteine at 65°C for 48 hr. The mixture was adjusted to pH 7.8 and then digested with trypsin I-EC 3.4.4.4] at 37°C for 72 hr with simultaneous dialysis against 0.1 M phosphate buffer of pH 7.8. The digested solution was deproteinized with 5% trichloroacetic acid in the cold
and the supernatant was dialyzed against several changes of cold deionized water for 72 hr. Then 4 vol of alcohol containing 1% acetic acid and 1% potassium acetate were added to precipitate the crude glycopeptide and glycosaminoglycan. The precipitate was washed with alcohol and ether, dried in a CaCI2 desiccator and then redissolved in 20 ml of deionized water. An aliquot was used for hexose, sialic acid and uronic acid analyses. Hexose was determined by the orcinol-sulfuric acid reaction of Winzler (1955); sialic acid by the diphenylamine method of Winzler (1955); and uronic acid by the carbazole reaction of Dische (1962), respectively. Cellulose acetate electrophoresis of the material was carried out using barbital buffer, pH 8.6,/~ = 0.06. Strips were run in duplicates for 30 min with a current of 1 mA per cm width. After electrophoresis, strips were stained with both alcian blue and PAS. Marker samples containing hyaluronic acid, heparan sulfate, chondroitin sulfate-A, chondroitin sulfate-B and chondroitin sulfate-C were applied alongside each gastric material; hyaluronic acid, chondroitin sulfate-A, -B and -C were purchased from Sigma (U.S.A.), and heparan sulfate was kindly given by Dr A. Cifonelli (Chicago University, U.S.A.), respectively. The species used were: Human, Homo sapiens; rabbit, Oryctolaffus cuniculus; horse, Equus caballus, cow, Bos taurus; pig, Sus scrofa; goat, Capra hircus; sheep, Ovis aries; dog, Canis familiaris; cat, Felis catus; guinea pig, Cavia cobaya; rat, Rattus norve#icus. RESULTS
AND DISCUSSION
Results of the hexose, sialic acid and uronic acid analyses of the crude glycopeptide and glycosaminoglycans from the human, equine, bovine and porcine gastric walls are summarized in Table 1. Both the hexose and sialic acid contents of the mucosae were higher in the porcine stomach than in the human and equine stomach§. Concerning both the hexose and sialic acid contents of the mucosae from bovine stomachs, the values were highest in the fourth stomach among four kinds of stomachs, and similar
163
164
H . MASUDA, S. SHICHIJO AND M. TAKEUCHI TABLE 1. GLYCOPEPTIDE AND GLYCOSAM]NOGLYCAN CONTENTS IN THE STOMACH OF SEVERAL ANIMALS*
Species
Layer
H u m a n stomach
Mucosa Muscular-serosa Mucosa Museular-serosa Mucosa Museular-serosa Mucosa Muscular-serosa Mucosa Muscular-serosa Mucosa Muscular-serosa Mucosa Muscular-serosa
Equine stomach Bovine first stomach Bovine second stomach Bovine third stomach Bovine fourth stomach Porcine stomach
Hexose 6.3 2.8 5.3 3.3 4.3 1.4 3.3 1.7 5.5 2.9 13.0 2.3 17.9 3.0
-I- 1.62 ___ 1.00 _ 1.21 _ 1.24 _ 0.69 + 0.61 + 0.92 _ 0.52 ± 1.65 + 0.18 + 4.67 _ 1.21 _ 0.63 + 0.60
Sialic acid 0.5 0.4 0.5 0.5 0.5 0.4 0.4 0.4 0.7 0.6 1.2 0.4 1.3 0.4
+ 0.09 _ 0.10 _ 0.08 + 0.07 + 0.15 + 0.21 + 0.23 + 0.14 _ 0.34 + 0.02 ___ 0.22 + 0.23 + 0.14 + 0.09
Uronic acid 0.6 0.6 1.0 0.8 0.8 0.7 0.6 0.6 1.1 0.9 1.7 0.8 1.5 0.6
+ 0.07 + 0.09 ± 0.17 + 0.12 + 0.17 + 0.15 + 0.15 + 0.18 + 0.68 + 0.04 _ 0.17]" ___ 0.23 _ 0.12t + 0.09
* Expressed as mg/1 g of dry tissue. t Although the uronic acid contents of the swine and the bovine fourth stomach mucosae were higher than another stomach, the carbazole reaction of the materials from these two mucosae gave a brown colour which was not typical of uronic acid, and so might probably have more uronic acid than correct content.
PAS stain
Alcian blue stain
5 4 5 6 7 HA
Serum Ponceau-S)
HS hS I
0
Anode
Anode
Fig. 1. Electrophoretic patterns of the glycopeptide and glycosaminoglycans from the gastric mucosae on cellulose acetate strip. 1. H u m a n stomach; 2. Equine stomach; 3. Bovine first stomach; 4. Bovine second stomach; 5. Bovine third stomach; 6. Bovine fourth stomach; 7. Porcine stomach; HA. Hyaluronic acid; HS. Heparan sulfate; ChS. A mixture of chondroitin sulfate-A, -B and -C.
Comparative studies on the gastric glycopeptide results were obtained by the similar studies on the stomachs of both goat and sheep. Cellulose acetate strip-electrophoresis of materials from the mucosae revealed the presence of one PAS positive and three alcianophilic components in human, equine and bovine first to third stomachs. The former moved slightly to the anode, and the latter components migrated rapidly toward the anode and were identified as hyaluronic acid, heparan sulfate and chondroitin sulfates, as reported previously (Kimoto et al., 1965). In both bovine fourth and porcine stomachs, another component was also found which stained with both PAS and alcian blue. It migrated toward the anode slower than" hyaluronic acid (Fig. 1). Stomach tissues of rabbit, horse, dog, cat, guinea pig, rat, goat and sheep were also studied similarly. The glycopeptide, which was stained with both PAS and alcian blue, were absent or sparingly present in the mucosae of the rabbit, horse and first to third stomachs of the goat and sheep, whereas in the mucosae of the dog, cat, guinea pig, rat and the fourth stomachs of the goat and sheep, it was found to noticeable extents. And furthermore, it was not observed in the gastric muscular-serosa of any animals of the eleven species tested. Hakkinen et al. (1965) and Slomiany et al. (1972) isolated, after proteolytic digestion, sulfated glycopeptides from the dog and porcine gastric mucosae, respectively. Further investigation will be necessary to determine how the
165
glycopeptide from gastric mucosa of each animal differs biochemically from those from different kinds of animals.
REFERENCES
DISCHE Z. (1962) Color reactions of carbohydrates. In Methods in Carbohydrate Chemistry. (Edited by WHISTLERR. L. H WOLFORMM. L.), Vol, 1, pp. 497-501. Academic Press, New York. HAKKINENI., HATIALAK. H TERHOT. (1965) The fractionation and characterization of the acid polysaccharides of the gastric wall of the dog. Acta chem. scand. 19, 800-806. HOUGH L. & JONES J. V. S. (1972) Human gastric mucosa part 1. The preparation of a glycopolypeptide and some aspects of its structure. Carbohydrate Res. 23, 1-16. KIMOTOE., TANAKAY., KURANARIT., MASUDAH. & TAKEtJCm M. 0965) Sialomucopolysaccharides of human cancerous stomach. Proc. Syrup. Chem. Physiol. 5, 122-128. SCHILLER S., SLOVER G. A. & DORFMANN A. (1961) A method for the separation of acid mucopolysaecharides; Its application to the isolation of heparin from the skin of rats. J. biol. Chem. 236, 983-990. SLOMIANYB. L. H MEYER K. (1972) Isolation and structural studies of sulfated glycoproteins of hog gastric mucosa. J. biol. Chem. 247, 5062-5070. WINZLER R. J. (1955) Determination of serum glycoproteins. In Methods of Biochemical Analysis (Edited by GLICK D.), Vol. 2. pp. 279-324. Interscience, New York.