Richard J. Winzler

Sulfonated amino sugars derived from alkaline sulfite-treated glycoproteins

Abstract A new compound has been found by ion exchange chromatography in hydrolysates of several alkaline sulfite-treated glycoproteins. The material was identified as a hexosamine sulfonic acid by comparison with material synthesized from N -acetyl-hexosamines. The sulfonic acid residue seems to be attached to carbon 3 of the amino sugar. The alkaline sulfite treatment of glycoproteins has been shown to be a useful procedure to discriminate between substitution at the carbon atom 4 as opposed to carbon atoms 3 and 6 on a hexosamine bound by an O -glycosidic linkage to the peptide chain.

Sequential elution of proteins in small volumes by preparative polyacrylamide gel electrophoresis

Abstract A simple flexible method for separation of proteins by polyacrylamide gel electrophoresis and sequential elution into dialysis bags has been devised. The system was applied to isolation of three glycoproteins from the peritoneal fluid of mice bearing Ehrlich ascites tumor.

Purification and chemical characterization of the major glycoprotein of avian myeloblastosis virus

Abstract The major glycoprotein of avian myeloblastosis virus (AMV) has been purified to an apparent state of homogeneity by gel filtration on a Sepharose 4B column in the presence of 6 m guanidine hydrochloride followed by dialysis against distilled water and then extraction with chloroform-methanol. The AMV glycoprotein remains soluble in the aqueous phase whereas contaminating proteins precipitate, either upon dialysis against distilled water or after treatment with chloroform-methanol. Carbohydrate, represented by glucosamine, mannose, galactose, fucose, and sialic acid, constitutes 40% of the weight of AMV glycoprotein. Glucosamine is the major carbohydrate component whereas fucose and sialic acid are present in relatively low amount. Amino acid analysis indicates a relatively high content of aspartic and glutamic acid, serine, threonine, and glycine. Based on SDS-polyacrylamide gel electrophoresis, a molecular weight value of 77,500 ± 500 was determined for AMV glycoprotein.

Characterization of monosaccharides in human placental alkaline phosphatase by gas—liquid chromatography

Abstract Gas—liquid chromatography of hydrolysates of highly purified human placental alkaline phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) demonstrated the presence of monosaccharide residues, mannose, galactose, glucose and fucose. This enzyme, therefore, is a sialoglycoprotein.

Stimulation of [3H]glucosamine incorporation into the cell surface of Amoeba by pinocytosis☆

Suspensions of amoebae were treated with [3H]glucosamine either before or after exposure to solutions of KCl or Alcian blue. Several hours later, the cell surface was isolated. Comparison of specific activities of control and experimental material showed that pinocytosis caused a specific increase in the amount of [3H]glucosamine incorporated into the cell surface fraction. Ion-exchange chromatography of acid hydrolysates of pooled radiolabeled surface fractions showed that all of the label eluted at positions corresponding to glucosamine and galactosamine.

Glycoproteins from ascitic fluid of Ehrlich ascites tumor: Isolation and chemical characterization

Three glycoprotein bands were identified by polyacrylamide disc gel electrophoresis in the perchloric acid soluble fraction of ascitic fluid of Ehrlich ascites tumor in mice. The three proteins were first separated by a new discontinuous preparative electrophoresis apparatus described previously [1]. They were further purified on Sephadex G-100 and then were subjected to chemical characterization. These glycoproteins were rich in glutamic and aspartic acids and contained the sugar moieties galactose, mannose, fucose, N-acetyl-D-glucosamine and sialic acid. The percent sugar composition ranged from 17.7-37.3% of the total weights of these glycoproteins.

GLYCOPROTEIN ANTIGENS ISOLATED FROM RBC

Abstract Erythrocytes contain a glycoprotein whose monomeric molecular weight is 31,000. This glycoprotein is highly assymetric with respect to the distribution of carbohydrate along the peptide chain and with respect to the distribution of serine, threonine and the amino acids with lipophilic side chains. At least two quite different types of oligosaccharide units are attached to the peptide chain by two different kinds of linkage. On the basis of the compositional studies a model is presented for the structure of the glycoprotein and its association with the erythrocyte membrane.

The identification of phosphorylated neo inositol as an anionic component of the amoeba cell surface

Abstract A nonreducing polyol, previously called pre-mannose [Allen et al. (1974) J. Cell Biol. 60 , 26–38], present in the isolated cell surface of Amoeba has been identified as neo inositol. The polyol was present on the cell surface as a highly phosphorylated but nonpolymerized phosphate ester. Cold acid extraction of Amoeba homogenates and alkaline precipitation of the acid-soluble fraction gave a preparation containing the polyol as the major neutral sugar residue. The polyol was identified by gas-liquid chromatography and mass spectrometry of its trimethylsilyl ether and acetate ester derivatives. The acid-labile phosphate ester present in isolated surface fractions was found to be separable from the acid-stable phosphate ester. Current data indicated that the labile phosphate was polyphosphate.

Physical studies on glycoproteins from ascitic fluid of ehrlich ascites tumor.

Three glycoproteins, designated as F, M and S glycoproteins were identified in the HCIO4-soluble fraction of ascitic fluid of Ehrlich ascites tumor by 8% polyacrylamide disc gel electrophoresis. They were separated and purified as described previously (Reznick, A.Z. and Winzler, R.J. (1973) Fed. Proc. 32, 368 and Reznick, A.Z., Allen, H.J. and Winzler, R.J. (1973) Anal. Biochem. 52, 395-401) and subjected to physical characterization. Several physical properties such as molecular weights, sedimentation and diffusion coefficients, partial specific volumes, Stokes radii and frictional ratios were determined. The physical parameters of F and S glycoproteins resemble data that have been reported for orosomucoid and haptoglobin-like glycoproteins, respectively. Properties of M glycoprotein could not be associated with a known glycoprotein.