Search

A glycoprotein (GP72) has been isolated from Trypanosoma cruzi and found to contain 41% protein, 49% carbohydrate and 10% phosphate. All phosphate was covalently attached to the carbohydrate which contained the following sugars: ribose, xylose, fucose, galactose, mannose, glucose and glucosamine. The carbohydrate side chains were linked to protein by fucose, xylose and N-acetylglucosamine; 50% of the total N-acetylglucosamine was involved in glycoprotein linkages. Two classes of carbohydrate side chains were detected. One class comprised 15% of the total carbohydrate and contained glucosamine, mannose and galactose; some of these chains were phosphorylated. The other class comprised 85% of the total carbohydrate and contained xylose, ribose, fucose, galactose, mannose, glucosamine and phosphate; these chains were antigenic and reacted with a monoclonal antibody with specificity for the whole glycoprotein.

Pig lymphocyte plasma membrane isolated from mesenteric lymph node contained 69 mug of carbohydrate/mg dry wt., which was made up of neutral sugar, amino sugar and sialic acid in the molar proportions 5:1.7:1. The neutral sugar comprised fucose, ribose, mannose, glucose, galactose and inositol (molar proportions 2:9:11:15:26:1), and the amino sugar glucosamine and galactosamine (molar ratio 2:1). The ribose was most probably derived from RNA. All of the fucose and mannose and almost all of the glucosamine were associated with the membrane protein whereas the membrane lipid contained all of the inositol. The remaining sugars were distributed in various ratios between the protein and lipid fractions.

1. Optimum conditions, including the effect of media of different pH values, were determined for the incorporation of radioactive precursors into mucoproteins by pig gastric mucosa in vitro. 2. Mucosal scrapings incorporated radioactivity from [U-14C]-glucose and from [G-3H]threonine or [G-3H]serine solely into the carbohydrate and protein portions respectively of the mucoprotein molecules. 3. Of the radioactive mucoprotein 22% was water-soluble and up to 80% of the remainder was soluble in other solvents. 4. Pronase was the most successful proteolytic enzyme tested for making the mucoprotein water-soluble, up to 94% dissolving after digestion. 5. The Pronase digestion products of the mucoproteins were separated from protein by equilibrium-density-gradient centrifugation in a CsCl gradient. 6. These Pronase-digested mucoproteins were further fractionated on Sepharose 4B and the isolated fractions analysed by chemical and sedimentation-velocity methods. 7. Pronase digestion and solvent extraction of mucosal scrapings labelled with 14C in the carbohydrate and 3H in the protein showed that one type of mucoprotein was the only non-diffusible biosynthetic product of the scrapings in vitro, and that this mucoprotein was the only mucoprotein constituent of the water-soluble and water-insoluble mucus.

1. Gel filtration of the water-soluble radioactive mucus produced three radioactive fractions, fraction A excluded on Sepharose 4B, fraction B included on Sepharose 4B but excluded on Sephadex G-200, and fraction C included on Sephadex G-200. 2. The specific radioactivities of fractions A and B were the same, with fraction C a little lower, whether the material was labelled with 14C-labelled carbohydrate or with 3H-labelled protein prepared by incubation of mucosal scrapings in vitro with [U-14C]glucose or [G-3H]threonine respectively. 3. Fractions A and B had an analysis of protein 22%, hexose 28%, hexosamine 28%, fucose 10% and sialic acid 1%; fraction C had an analysis closely similar to this, except that it contained about 10% of a protein contaminant. 4. All three fractions had closely similar A and H blood-group activities. 5. Ultracentrifuge studies showed fractions A, B and C were polydisperse with s025,w values of 18.7S, 4.9S and 3.9S respectively. 6. The unfractionated water-soluble mucus contained only two peaks, fraction A 18.7S and a peak of 4.4S, which was a combination of fractions B and C. 7. The radioactive mucoprotein accounted for 85% by weight of the soluble mucus and the results show that it consisted of two distinct fractions A and B–C, which were chemically, biosynthetically and immunologically very similar.