Enzymatic sulfation of mucus glycoprotein in gastric mucosa. Effect of ethanol

S. R. Carter, Amalia Slomiany, K. Gwozdzinski, Y. H. Liau, Bronislaw Slomiany

Research output: Contribution to journalArticle

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Abstract

A sulfotransferase activity that catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate to carbohydrate chains of gastric mucus glycoprotein has been demonstrated in the antral and body mucosa of rat stomach. Subcellular fractionation studies revealed that the enzyme is associated with Golgi-rich membrane fraction. The sulfotransferase activity of this fraction in antral mucosa was about 35% lower than that of the body. Optimum enzyme activity was obtained with 0.5% Triton X-100 and 30 mM NaF at a pH of 6.8 using desulfated mucus glycoprotein substrate. The enzyme was equally capable of sulfation of the proteolytically degraded and reduced forms of the desulfated glycoprotein, but the acceptor capacity of the intact mucus glycoprotein was about 60% lower than that of the desulfated preparation. The enzyme preparation also catalyzed the transfer of sulfate to galactosylceramide. The sulfation of mucus glycoprotein, however, was not affected by the presence of this glycolipid, suggesting that the sulfotransferase involved in mucus glycoprotein sulfation is different from that responsible for the synthesis of sulfatoglycosphingolipid. The mucus glycoprotein sulfotransferase activity was inhibited by ethanol. The rate of inhibition was proportional to the concentration of ethanol up to 0.3 M and was of the competitive type. The apparent K(m) value of the enzyme for mucus glycoprotein was 10.5 x 10-6 M (21 mg/ml), and the K(I) in the presence of ethanol was 4.7 x 10-1 M. The 35S-labeled mucus glycoprotein product of the enzyme reaction gave in CsCl density gradient a band in which the 35S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein led to the liberation of the label into reduced acidic oligosaccharide fraction. Most of the label was found incorporated in three oligosaccharides. These were identified as tri-, tetra-, and pentasaccharides, each carrying a labeled sulfate ester group on the terminal N-acetylglucosamine residue. Based on the results of structural analyses, the most abundant oligosaccharide was characterized as SO3H→6GlcNAcβ1→3Galβ1→3GalNAc-ol.

Original languageEnglish (US)
Pages (from-to)11977-11984
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number24
StatePublished - Jan 1 1988

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Gastric Mucosa
Ethanol
Enzymes
Oligosaccharides
Sulfates
Labels
Sulfotransferases
Glycoproteins
galactosylceramide sulfotransferase
Esters
Mucous Membrane
Phosphoadenosine Phosphosulfate
Galactosylceramides
Sulfoglycosphingolipids
Borohydrides
Acetylglucosamine
Octoxynol
Enzyme activity
Fractionation
gastric mucus glycoproteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{b42f873e1c9c4466a012d300e0cd9b11,
title = "Enzymatic sulfation of mucus glycoprotein in gastric mucosa. Effect of ethanol",
abstract = "A sulfotransferase activity that catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate to carbohydrate chains of gastric mucus glycoprotein has been demonstrated in the antral and body mucosa of rat stomach. Subcellular fractionation studies revealed that the enzyme is associated with Golgi-rich membrane fraction. The sulfotransferase activity of this fraction in antral mucosa was about 35{\%} lower than that of the body. Optimum enzyme activity was obtained with 0.5{\%} Triton X-100 and 30 mM NaF at a pH of 6.8 using desulfated mucus glycoprotein substrate. The enzyme was equally capable of sulfation of the proteolytically degraded and reduced forms of the desulfated glycoprotein, but the acceptor capacity of the intact mucus glycoprotein was about 60{\%} lower than that of the desulfated preparation. The enzyme preparation also catalyzed the transfer of sulfate to galactosylceramide. The sulfation of mucus glycoprotein, however, was not affected by the presence of this glycolipid, suggesting that the sulfotransferase involved in mucus glycoprotein sulfation is different from that responsible for the synthesis of sulfatoglycosphingolipid. The mucus glycoprotein sulfotransferase activity was inhibited by ethanol. The rate of inhibition was proportional to the concentration of ethanol up to 0.3 M and was of the competitive type. The apparent K(m) value of the enzyme for mucus glycoprotein was 10.5 x 10-6 M (21 mg/ml), and the K(I) in the presence of ethanol was 4.7 x 10-1 M. The 35S-labeled mucus glycoprotein product of the enzyme reaction gave in CsCl density gradient a band in which the 35S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein led to the liberation of the label into reduced acidic oligosaccharide fraction. Most of the label was found incorporated in three oligosaccharides. These were identified as tri-, tetra-, and pentasaccharides, each carrying a labeled sulfate ester group on the terminal N-acetylglucosamine residue. Based on the results of structural analyses, the most abundant oligosaccharide was characterized as SO3H→6GlcNAcβ1→3Galβ1→3GalNAc-ol.",
author = "Carter, {S. R.} and Amalia Slomiany and K. Gwozdzinski and Liau, {Y. H.} and Bronislaw Slomiany",
year = "1988",
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volume = "263",
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Enzymatic sulfation of mucus glycoprotein in gastric mucosa. Effect of ethanol. / Carter, S. R.; Slomiany, Amalia; Gwozdzinski, K.; Liau, Y. H.; Slomiany, Bronislaw.

In: Journal of Biological Chemistry, Vol. 263, No. 24, 01.01.1988, p. 11977-11984.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Enzymatic sulfation of mucus glycoprotein in gastric mucosa. Effect of ethanol

AU - Carter, S. R.

AU - Slomiany, Amalia

AU - Gwozdzinski, K.

AU - Liau, Y. H.

AU - Slomiany, Bronislaw

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Y1 - 1988/1/1

N2 - A sulfotransferase activity that catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate to carbohydrate chains of gastric mucus glycoprotein has been demonstrated in the antral and body mucosa of rat stomach. Subcellular fractionation studies revealed that the enzyme is associated with Golgi-rich membrane fraction. The sulfotransferase activity of this fraction in antral mucosa was about 35% lower than that of the body. Optimum enzyme activity was obtained with 0.5% Triton X-100 and 30 mM NaF at a pH of 6.8 using desulfated mucus glycoprotein substrate. The enzyme was equally capable of sulfation of the proteolytically degraded and reduced forms of the desulfated glycoprotein, but the acceptor capacity of the intact mucus glycoprotein was about 60% lower than that of the desulfated preparation. The enzyme preparation also catalyzed the transfer of sulfate to galactosylceramide. The sulfation of mucus glycoprotein, however, was not affected by the presence of this glycolipid, suggesting that the sulfotransferase involved in mucus glycoprotein sulfation is different from that responsible for the synthesis of sulfatoglycosphingolipid. The mucus glycoprotein sulfotransferase activity was inhibited by ethanol. The rate of inhibition was proportional to the concentration of ethanol up to 0.3 M and was of the competitive type. The apparent K(m) value of the enzyme for mucus glycoprotein was 10.5 x 10-6 M (21 mg/ml), and the K(I) in the presence of ethanol was 4.7 x 10-1 M. The 35S-labeled mucus glycoprotein product of the enzyme reaction gave in CsCl density gradient a band in which the 35S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein led to the liberation of the label into reduced acidic oligosaccharide fraction. Most of the label was found incorporated in three oligosaccharides. These were identified as tri-, tetra-, and pentasaccharides, each carrying a labeled sulfate ester group on the terminal N-acetylglucosamine residue. Based on the results of structural analyses, the most abundant oligosaccharide was characterized as SO3H→6GlcNAcβ1→3Galβ1→3GalNAc-ol.

AB - A sulfotransferase activity that catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate to carbohydrate chains of gastric mucus glycoprotein has been demonstrated in the antral and body mucosa of rat stomach. Subcellular fractionation studies revealed that the enzyme is associated with Golgi-rich membrane fraction. The sulfotransferase activity of this fraction in antral mucosa was about 35% lower than that of the body. Optimum enzyme activity was obtained with 0.5% Triton X-100 and 30 mM NaF at a pH of 6.8 using desulfated mucus glycoprotein substrate. The enzyme was equally capable of sulfation of the proteolytically degraded and reduced forms of the desulfated glycoprotein, but the acceptor capacity of the intact mucus glycoprotein was about 60% lower than that of the desulfated preparation. The enzyme preparation also catalyzed the transfer of sulfate to galactosylceramide. The sulfation of mucus glycoprotein, however, was not affected by the presence of this glycolipid, suggesting that the sulfotransferase involved in mucus glycoprotein sulfation is different from that responsible for the synthesis of sulfatoglycosphingolipid. The mucus glycoprotein sulfotransferase activity was inhibited by ethanol. The rate of inhibition was proportional to the concentration of ethanol up to 0.3 M and was of the competitive type. The apparent K(m) value of the enzyme for mucus glycoprotein was 10.5 x 10-6 M (21 mg/ml), and the K(I) in the presence of ethanol was 4.7 x 10-1 M. The 35S-labeled mucus glycoprotein product of the enzyme reaction gave in CsCl density gradient a band in which the 35S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein led to the liberation of the label into reduced acidic oligosaccharide fraction. Most of the label was found incorporated in three oligosaccharides. These were identified as tri-, tetra-, and pentasaccharides, each carrying a labeled sulfate ester group on the terminal N-acetylglucosamine residue. Based on the results of structural analyses, the most abundant oligosaccharide was characterized as SO3H→6GlcNAcβ1→3Galβ1→3GalNAc-ol.

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