Evidence for the occurrence of lactosaminoglycan in porcine kidney
Tohoku journal of experimental medicine
Weakly acidic glycopeptides were obtained from the pronase digest of porcine kidney. The glycopeptides were digested with endo -f3-galactosidase. An oligosaccharide fraction was separated from the digest by gel-filtration through Sephadex G-50. The fraction contained galactose and glucosamine in an equimolar ratio. lactosaminoglycan ; porcine kidney Lactosaminoglycans have been shown to be present on cell surfaces of various cell types and are thought to be one of the differentiation antigens
... ntiation antigens (Spooncer and Fukuda 1984) . In a previous work, we isolated weakly acidic glycopeptide fraction from porcine kidney after pronase digestion (Munakata et al. 1985) . The results of the f3-elimination reaction indicated the presence of glycopeptides derived from mucin-type glycoproteins, but it was also shown that these fractions might contain glycopeptides derived from glycoproteins other than those of mucin-type (Munakata et al. 1985) . These glycopeptides contained galactose and N-acetylglucosamine as major constituents. It is, therefore, of interest to examine whether lactosaminoglycan is present in the kidney glycopeptide fractions. Weakly acidic glycopeptides (Munakata et al. 1985) were further separated by gelfiltration on Sephadex G-100 from mucin-type glycopeptides which were eluted at the void volume from the column. From 1,000 g of wet kidneys, 50 mg of purified glycopeptides were obtained. The glycopeptides were incubated with Escherichia freundii endo-R-galactosidase (Seikagaku Kogyo Co.) in 0.2 M sodium acetate buffer (pH 5.8) at 37°C for 24 hr (Li et al. 1982) . The digest was chromatographed on a column of Sephadex G-50 (Fig. 1) . The results indicated that the glycopeptides contained the carbohydrate moieties sensitive to endo-f-galactosidase which cleaves f3-galactosidic linkages of lactosaminoglycan into a disaccharide having structure of N-acetylglucosamine (/1-3) galactose. A highly retarded fraction was then subjected to chromatography on a column of Cellulofine GCL-25-m. An oligosaccharide, which was positive to the phenol-H2SO4 reaction, was eluted at the position immediately after the elution position of glucose tetramer. Chemical analysis (Munakata et al. 1985) indicated that an oligosaccharide was composed of hexosamine and hexose in an equimolar ratio. This oligosaccharide was hydrolyzed with 2 M HCl at 100°C for 6 hr and the hydrolyzate was subjected to paper chromatography in butanol-pyridine-water (6: 4 : 3, v/v). Galactose and glucosamine were solely detected by the silver-nitrate staining. These results indicate that the material released by digestion with endo-Q-galactosidase is a disaccharide containing galactose and N-acetylglucosamine.