concanavalin-a and sodium-borohydride

A secreted glycoprotein (GP) with apparent molecular mass of 90 kDa produced by cultured embryonic cells of Drosophila melanogaster was isolated and partially characterized. GP is enriched by Ser + Thr and Pro residues that constitute up to 30% of the total number of amino acids. An abundant carbohydrate moiety (40% of molecular mass) is mainly represented by vertebrate mucin-type O-linked disaccharide units Gal(beta 1-3)-GalNAc, occupying about a half of the total number of Ser+Thr residues and rendering the GP molecule high resistance to protease action. A few of N-glycans are also present in GP. These characteristics allow to consider the Drosophila GP (termed 'mucin-D') as a first representative of invertebrate mucin-type glycoproteins.

Topics: Amidohydrolases; Amino Acids; Animals; Blotting, Western; Borohydrides; Carbohydrate Sequence; Carbohydrates; Cells, Cultured; Chemical Fractionation; Concanavalin A; Drosophila melanogaster; Embryo, Nonmammalian; Glycosylation; Hexosaminidases; Molecular Sequence Data; Mucins; Oligosaccharides; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Tunicamycin

Starches, isolated from rapidly expanding tobacco leaves four times during the day and night and once from fully expanded leaves, were fractionated with concanavalin A. From an examination of the amounts and properties of amylose, the diurnal decrease in iodine absorption of the starches on illumination appeared to be due to an increase in its branched character, and possibly the presence of unbranched polymer of low dp, combined with a decrease in the proportion of amylose fraction. The increase in apparent amylose content with age was due to an increase in the proportion of amylose. The amylose fractions at different times had only small differences in average mol size in solution and relative mol wt (M(r) near 4 x 10(5)) which were lower than those of storage organs. The average mol size in solution and relative mol wt of the amylopectins decreased during illumination, increased in darkness, and were lower (M(r) 2-2.7 x 10(6)) at all times than those from storage organs. Debranching followed by size-exclusion chromatography [on Fractogel TSK 50(S)] gave similar proportions of long, medium, and short chains for all amylopectin samples, and these proportions differed from those for debranched amylopectin from n-maize seed starch. On debranching and chromatography of the amylopectin beta-limit dextrins (which gives an estimate of the proportions of core chains) differences persisted. Structural characteristics of amylopectin from tobacco leaf starch were similar to those of normal genotypes from storage organs. The proportion of glucosyl units in core chains, the external-to-core chain ratio, and indices of compactness were calculated for a number of (1-->4)(1-->6) alpha-glucans. A plot of the index of compactness for glycogens and amylopectins showed that the decrease in compactness and the increase in total average chain length that occurs from glycogen to normal and then to amylose extender amylopectins involves a proportionate increase in average internal, external, and core chain lengths and not a selective increase in one type of chain.

Topics: Amylopectin; Amylose; Borohydrides; Cell Fractionation; Chromatography, Gel; Circadian Rhythm; Concanavalin A; Dextrins; Nicotiana; Periodic Acid; Plant Lectins; Plants, Toxic; Starch

Lactotransferrin isolated from a pool of mature bovine milk has been shown to contain N-glycosidically-linked glycans possessing N-acetylneuraminic acid, galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. The glycopeptides obtained by Pronase digestion were fractionated by concanavalin A-Sepharose affinity chromatography into three fractions: slightly retained (A), retained (B), and strongly retained (C). The structure of the glycans of the three fractions has been determined by application of methanolysis, methylation analysis, fast atom bombardment-mass spectrometry, and 1H NMR spectroscopy. Diantennary structures without GalNAc were present as partially sialylated and partially (1-->6)-alpha-L-fucosylated structures in Fractions A and B. Sequences containing alpha-D-Galp-(1-->3)-beta-D-Gal on the alpha-D-Man-(1-->6) antenna, and beta-D-GalpNAc-(1-->4)-beta-D-GlcNAc and alpha-NeuAc-(2-->6)-beta-D-GalpNAc-(1-->4)-beta-D-GlcNAc on the alpha-D-Man-(1-->3) antenna were characterized in the oligosaccharide-alditols obtained by reductive cleavage of Fraction B. A series of Man4-9-GlcNAc structures were identified in Fraction C after endo-N-acetyl-beta-D-glucosaminidase digestion. These results show that the structures of bovine lactotransferrin glycans are more heterogeneous than those of previously characterized transferrin glycans.

Topics: Animals; Borohydrides; Cattle; Chemical Fractionation; Concanavalin A; Glycopeptides; Lactoferrin; Magnetic Resonance Spectroscopy; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Methane; Methylation; Oligosaccharides; Polysaccharides; Protons; Spectrometry, Mass, Fast Atom Bombardment; Sugar Alcohols

Sialic acid residues of plasma membrane glycoproteins of rabbit corneal endothelial cells were radiolabeled by oxidation with sodium periodate and reduction with sodium borotritide. Surface-labeled glycoproteins were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The major surface labeled glycoproteins were designated GP 1-8 in order of their increasing mobility on the gel (M.W. = 220K (GP-1), 200K (GP-2), 170K (GP-3), 135K (GP-4), 110K (GP-5), 95K (GP-6), 80K (GP-7), and 44K (GP-8). On the basis of the behavior of these glycoproteins on various carrier-bound lectins, preliminary information concerning their saccharide moieties was obtained. All 8 components bound to agarose-linked wheat germ agglutinin; GP 4-6 bound to concanavalin A and GP 6-7 bound to Ricinus communis agglutinin. No component bound to Bandeiraea simplicifolia I, Bandeiraea simplicifolia II, Ulex europeus or soybean agglutinin. These data suggest that in addition to the presence of sialic acid/N-acetylglucosamine residues in all the eight glycoproteins, oligosaccharides with terminal beta-galactose residues occur in GP-6 and GP-7 while mannose (glucose) residues occur in GP 4-6.

Topics: Animals; Borohydrides; Chromatography, Affinity; Concanavalin A; Cornea; Culture Techniques; Electrophoresis, Polyacrylamide Gel; Endothelium; Lectins; Membrane Proteins; Periodic Acid; Rabbits; Sialoglycoproteins

Two fractions of herpes simplex virus glycoprotein gC were isolated and characterized by means of immunosorbent-purification with monoclonal antibodies against gC and Helix pomatia lectin (HPA) affinity chromatography. About 25 per cent of the glycoprotein gC population demonstrated affinity for the lectin, compatible with presence of N-acetylgalactosamine as terminal sugar of the oligosaccharide. The HPA-binding populations of gC appeared as two electrophoretic bands with lower molecular weights than the non-binding gC. The gC subfraction without affinity for the HPA was subjected to treatments aiming to desialylize the carbohydrate moiety. Only 5 per cent of the initially non-reactive fraction of gC became reactive to HPA after the treatments, suggesting that masking of penultimate N-acetylgalactosamine by sialic acid was not a main reason for lack of HPA affinity. Results of treatment with alkaline Na BH4 demonstrated presence of oligosaccharide-peptide linkages sensitive to beta-elimination suggesting O-glycosidic type of linkage. The subfraction of gC demonstrating affinity for HPA as well as gC devoid of HPA binding capacity both revealed affinity for Con A. Therefore N-glycosidically as well as O-glycosidically linked oligosaccharides seemed to be present on the one and same glycoprotein. On the basis of the results presented we assume that the glycosylation of HSV glycoprotein gC may lead to, at least, two populations of the glycoprotein gC, one with terminal N-acetylgalactosamine residues of oligosaccharides O-glycosidically linked to the polypeptide and the other without affinity for HPA. However, both populations of gC contain similar proportions of oligosaccharides of the high mannose or complex types with N-glycosidic carbohydrate-peptide linkages as indicated by their affinity for Con A.

Topics: Acetylgalactosamine; Borohydrides; Chemical Phenomena; Chemistry; Chromatography, Affinity; Concanavalin A; Galactosamine; Glycoproteins; Helix, Snails; Lectins; Simplexvirus; Viral Proteins