Studies on Glycopeptides Released by Trysin from Intact Human Erythrocytes<sup>*</sup>

Winzler, Richard J.; Harris, Edward D.; Pekas, Darold J.; Johnson, Clarence A.; Weber, Peter

doi:10.1021/bi00859a042

Cited by 191 publications

(60 citation statements)

References 33 publications

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“…It has been found earlier by Romanowska and Baranowski The release of carbohydrates during the alkaline reduction of M and N' glycoproteins and glycopeptides was accompanied by the release of amino acid components in a similar proportion. This finding is contrary to the results of Winzler et al [8] and Kathan and Adamany [ 7 ] , but the alkali-lability of some peptide components has been already observed in the course of hydrolysis of M and N substances with triethylamine [24] : in this procedure after three successive hydrolyses the degradation was completed and about a half of the material became diffusible, sugar and amino acid components being released in a similar degree. The alkali-sensitivity of some bonds linking amino acids is not clear ; it may be the result of the influence of sugar substituents on the particular peptide bonds or some amino acids may be involved in other than peptide bonds, e. g. alkalilabile ester linkages.…”

Section: Discussioncontrasting

confidence: 89%

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The Degradation of M and N Blood Group Glycoproteins and Glycopeptides with Alkaline Borohydride

Lisowska

1969

Eur J Biochem

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The sialic acid-free M and N substances and glycopeptides from their pronase digest were treated with alkaline borohydride and products of degradation were fractionated. Galactose and N-acetylgalactosamine were split off during the degradation in the form of disaccharide : galactosyl-N-acetylgalactosaminitol, and it was accompanied by the release of amino acid components. The conclusion has been drawn that a part of carbohydrate moiety of M and N substances represents trisaccharides: N-acetylneuraminyl-galactosyl-N-acetylgalactosamine, linked to serine and threonine residues in the peptide chain by an alkali-labile 0-glycosidic bond. The other type of oligosaccharides present in M and N substances is linked to peptide chain by an alkali-stabile bond and includes mannose, fucose, N-acetylglucosamine, the rest of sialic acid and galactose.The degraded M' and N' glycoproteins (M and N glycoproteins free of N-acetylneuraminic acid) did not inhibit the hemagglutination by Vicia graminea phytoagglutinins. The degraded native M and N substances were inactive towards both anti-M and anti-N rabbit immune sera, and anti-N phytoagglutinins from V . graminea, but they were still as good inhibitors of viral hemagglutination as untreated substances.The effect of alkali or alkaline borohydride on glycoproteins and mucopolysaccharides has been widely studied and it has been shown that serine and threonine are destroyed if they are linked to carbohydrate by an 0-glycosidic linkage involving the anomeric group of a sugar and the hydroxyl group of the amino acid. When alkaline treatment is carried out in the presence of sodium borohydride, the unsaturated amino acids formed from the serine and threonine during the p-elimination reaction are in part reduced to alanine and a-aminobutyric acid, respectively; the sugar involved in the 0-glycosidic bond is reduced to the corresponding alcohol [i-5] pronase fragments, the fractionation of products obtained and their partial identification. The part of this study was reported a t the 6th FEBS Meeting in Madrid, 1969. MATERIALS AND METHODS MaterialsThe M, N and MN blood group substances were prepared from human erythrocytes of group 0 by phenol extraction of ghosts [9]. Sialic acid was removed from isolated substances by hydrolysis with 0.05N H,SO, a t 80" for 60min, followed by neutralization with barium hydroxide, dialysis of the supernatant and lyophilization of the dialysate ; sialic acid-free preparations are referred to as M , N', and MN'. The sialic acid-free glycopeptides were obtained by pronase digestion of M , N', and MPU" blood group substances as described earlier [lo].Sephadex 6-25 was a product of Pharmacia (Uppsala), Bio-Gel P-6 and P-2 were produced by Calbiochem ; Cellulose MN for thin-layer chromatography was a product of Macherey-Nagel Co. Colorimetric MethodsNeutral sugars were determined by the phenolsulfuric acid method [ i l l , specific determination of

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Section: Discussioncontrasting

confidence: 89%

“…This approach has been extended to M and N blood group substances [6,7] and their trypsin fragments [8]. Treatment of M and N glycoproteins with alkali or alkaline borohydride results in a loss of serine, threonine and galactosamine that strongly supports the view that galactosamine is linked glycosidally to hydroxy-amino acids.…”

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confidence: 90%

The Degradation of M and N Blood Group Glycoproteins and Glycopeptides with Alkaline Borohydride

Lisowska

1969

Eur J Biochem

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“…It is obvious that the sialoylglycoprotein in red cell membrane preparations is an authentic membrane protein, since it contains the A, B, and NM blood-group antigens found on the cell surface. Labeling studies showed that one other protein (molecular weight 100,000) is found at the cell surface (3,(8)(9)(10)(11)(12)). Bretscher showed further that different parts of these proteins are labeled by chemical reagents that act from the outside or the inside of the membrane (13, 14); hence, these proteins appear to span the membrane and must be considered authentic membrane proteins.…”

Section: Proteins Synthesized By Intact Reticulocytesmentioning

confidence: 99%

“…There exist 7-9 main components (1-8); the major species include two polypeptides of molecular weight greater than 200,000 (4), a protein of molecular weight 100,000, and a sialoylgylcoprotein which contains the MN, A, and B blood-group activities (8). The latter two species are the only membrane proteins found on the cell surface (3,(9)(10)(11)(12); they may also penetrate to the interior surface of the membrane (13)(14)(15). Little is known, however, about the biosynthesis of erythrocyte membrane proteins: the types of erythropoetic cells that synthesize each protein, the type of polysome (free or membrane-attached) that synthesizes the proteins, and the factors that regulate their synthesis.…”

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confidence: 99%

Biosynthesis of Reticulocyte Membrane Proteins by Membrane-Free Polyribosomes

Lodish

1973

Proc. Natl. Acad. Sci. U.S.A.

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Intact rabbit reticulocyte cells synthesize two predominant species of membrane polypeptides; at least 10 reticulocyte membrane polypeptide species are not produced by the cells. Cell-free extracts of reticulocytes, free of any membranes or membrane-bound polyribosomes, synthesize large amounts of these two membrane polypeptides; one of these polypeptides appears to be modified, probably by loss of 20-40 amino acids, after it is incorporated into the membrane. Deprivation of hemin results in inhibition of synthesis by lysates of membrane proteins, globin, and all other cytoplasmic proteins.The protein composition of mammalian erythrocyte membrane is now fairly well established. There exist 7-9 main components (1-8); the major species include two polypeptides of molecular weight greater than 200,000 (4), a protein of molecular weight 100,000, and a sialoylgylcoprotein which contains the MN, A, and B blood-group activities (8). The latter two species are the only membrane proteins found on the cell surface (3, 9-12); they may also penetrate to the interior surface of the membrane (13-15). Little is known, however, about the biosynthesis of erythrocyte membrane proteins: the types of erythropoetic cells that synthesize each protein, the type of polysome (free or membrane-attached) that synthesizes the proteins, and the factors that regulate their synthesis.The reticulocyte is the penultimate cell in the erythropoetic series; it has no nucleus, and makes no RNA or DNA.About 90% of protein produced is a and ,3-globin chains (16,17). The protein composition of reticulocyte membranes is very similar to that of erythrocyte membranes (my unpublished observations). In this paper, I show that reticulocytes synthesize only 2 major and 2-3 minor membrane proteins; most of the membrane proteins are no longer being synthesized in significant amounts at this stage.In most mammalian cells a fraction of the polyribosomes are firmly attached to membranes; these are the site of synthesis of most, if not all, proteins that are eventually transported out of the cell, such as trypsinogen, chymotrypsinogen, albumin, and other serum proteins (18-22). It is not at all clear that this is the sole function of these polysomes.Burka has claimed that rabbit reticulocytes contain membrane-bound polyribosomes (23) that synthesize most of the nonglobin proteins made by the cell (24). By contrast, we showed recently that membrane-free reticulocyte polyribosomes will produce precisely the same globin and nonglobin proteins as does the intact cell (17). In this paper I show, using dodecyl sulfate-gel electrophoresis and peptide map-1526 ping procedures, that a membrane-free lysate makes large amounts of the two membrane polypeptides produced by the intact cell; one of these proteins appears to be modified, probably by removal of 20-40 amino acids when it is incorporated into the membrane. MATERIALS AND METHODSMaterials. Acetylphenylhydrazine was purchased from Sigma, and [35S]methionine (30,000 Ci/mol) from Amersham/Searle. Sources of all o...

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“…Glycoproteins and glycolipids have been shown to be present in the plasma membranes of many mammalian cells including erythrocytes (1)(2)(3)(4)(5)(6)(7)(8)(9). Although numerous biological roles have been ascribed to these substances such as ABO and MN blood group antigenicity (3)(4)(5)(6)(7)(8), ability to inhibt influenza virus hemagglutination reacReceived for publication 18 January 1972 and in revised form 6 March 1972. tions (9), tumor antigenicity (10)(11), ion and macromolecular transport (12), and control of cellular adhesion (13,14), little is known about their chemistry and biosynthesis.…”

Section: Introductionmentioning

confidence: 99%

Glycosyltransferases in human blood

Kim¹,

Perdomo²,

Whitehead³

1972

J. Clin. Invest.

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A B S T R A C T Human serum and hemoglobin-free erythrocyte membranes were found to contain a galactosyltransferase which catalyzes the transfer of galactose from UDP-galactose to specific large and small molecular weight acceptors. The requirements for enzyme activity were found to be similar for the enzymes from both sources. However, the membrane-bound enzyme depended on a detergent for maximal activity. Mn++ was an absolute requirement for transfer and uridine nucleoside phosphates were inhibitors. The most effective acceptor for galactose was a glycoprotein containing N-acetylglucosamine residues in the terminal position of its oligosaccharide side chains. N-acetylglucosamine was also an acceptor. While the presence of a-lactalbumin in the incubation medium resulted in a significant decrease in the transfer of galactose to N-acetylglucosaxmine, glucose, which was not an acceptor for galactose in the absence of a-lactalbumin, became an excellent acceptor. The serum enzyme catalyzed the transfer of 54 nmoles of galactose per milliliter of serum per hour and its apparent Km for UDP-galactose was 7.5 X 10'M. The membrane enzyme had a similar apparent Km. Using a quantitative assay system the enzyme was found to be present in all individuals studied, regardless of their blood type, secretor status, or sex.

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Studies on Glycopeptides Released by Trysin from Intact Human Erythrocytes^*

Cited by 191 publications

References 33 publications

The Degradation of M and N Blood Group Glycoproteins and Glycopeptides with Alkaline Borohydride

The Degradation of M and N Blood Group Glycoproteins and Glycopeptides with Alkaline Borohydride

Biosynthesis of Reticulocyte Membrane Proteins by Membrane-Free Polyribosomes

Glycosyltransferases in human blood

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Studies on Glycopeptides Released by Trysin from Intact Human Erythrocytes*

Cited by 191 publications

References 33 publications

The Degradation of M and N Blood Group Glycoproteins and Glycopeptides with Alkaline Borohydride

The Degradation of M and N Blood Group Glycoproteins and Glycopeptides with Alkaline Borohydride

Biosynthesis of Reticulocyte Membrane Proteins by Membrane-Free Polyribosomes

Glycosyltransferases in human blood

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Product

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Studies on Glycopeptides Released by Trysin from Intact Human Erythrocytes^*