Preparation Of Glycopeptides Research Articles

Comparison of glycopeptide in lung cancer tissues of different histological types.

The glycopeptide was studied in the pulmonary cancer tissues of three different histological types including squamous cell carcinoma, undifferentiated cell carcinoma, and adenocarcinoma. The glycopeptide preparation was made by digesting the lung cancer tissues with proteolytic enzymes followed by removal of enzymes and undigested protein. The comparison of the relative proportion of the glycopeptide, which migrated toward the anode slower than hyaluronic acid, and was stained with both alcian blue and PAS, and contained higher sialic acid, was carried out among three different histologic types of lung cancer. This glycopeptide was highest in adenocarcinoma, followed by undifferentiated cell carcinoma, and was lowest in squamous cell carcinoma.

Solid Phase Synthesis of Peptides and Glycopeptides on Polymeric Supports with Allylic Anchor Groups

New polymeric supports (Hycram resins) have enabled the preparation of sensitive glycopeptides by solid-phase synthesis. These resins make it possible to work with acid- and base-labile protective groups, since the glycopeptides and peptides thus prepared can be recovered from the support by palladium(0) catalysis under neutral, weakly basic or weakly acid conditions. Acid(Boc)-labile and base-labile protective groups remain intact. Examples are the synthesis of the glycotripeptides 1b and of the Leu-enkephalin 2b protected at the tyrosine via the Hycram compounds 1a and 1b.

The covalent structure of individual N-linked glycopeptides from ovomucoid and asialofetuin.

In order to explore whether individual N-linked glycans in a given glycoprotein may be processed to different end products and at the same time prepare a number of well characterized glycopeptides as substrates for glycopeptide hydrolases, we have prepared the individual glycopeptides representing the four major glycosylation sites in ovomucoid and the three sites in asialofetuin. The individual glycopeptides were characterized by amino acid sequence determination before and after removal of the glycan by peptide:N-glycanase (amidase), and the liberated glycans were subjected to mass spectrometric analysis. As expected from available sugar analyses of the individual glycans in ovomucoid, no major differences were detected between the four glycosylation sites in this glycoprotein, but a definite trend toward less processed (less extensively branched) species was observed in going from site 1 to 4. In fetuin, for which the glycan pool is known to be made up of about two-thirds triantennary and one-third biantennary structures, the analysis of the three glycopeptides gave triantennary to biantennary ratios of 75/25, 67/33, and 70/30, respectively, demonstrating that the three sites are processed to a very similar, albeit perhaps not identical, extent. All the glycopeptides obtained in these studies, including the CNBr-produced glycopeptide from ovalbumin, were purified by a set series of steps, gel filtration on Sephadex G-50 followed by ion-exchange chromatography on DE52 and/or reverse phase high performance liquid chromatography. Based on the results, these procedures appear to have general application for the preparation of glycopeptides.

Characterization of a novel linkage unit between ribitol teichoic acid and peptidoglycan in Listeria monocytogenes cell walls.

The structure of the linkage unit between ribitol teichoic acid and peptidoglycan in the cell walls of Listeria monocytogenes EGD was studied. A teichoic-acid--glycopeptide preparation isolated from lysozyme digests of the cell walls of this strain contained mannosamine, glycerol, glucose and muramic acid 6-phosphate in an approximate molar ratio of 1:1:2:1, together with large amounts of glucosamine and other components of teichoic acid and glycopeptides. A teichoic-acid-linked sugar preparation, obtained by heating the cell walls at pH 2.5, also contained glucosamine, mannosamine, glycerol and glucose in an approximate molar ratio of 25:1:1:2. Part of the glucosamine residues were shown to be involved in the linkage unit. Thus, on mild alkaline hydrolysis, the teichoic-acid-linked sugar preparation gave a disaccharide characterized as N-acetylmannosaminyl(beta 1----4)-N-acetylglucosamine [ManNAc(beta 1----4)GlcNAc] in addition to the ribitol teichoic acid moiety, whereas the teichoic-acid - glycopeptide was separated into disaccharide-linked glycopeptide and the ribitol teichoic acid moiety by the same procedure. Furthermore, Smith degradation of the cell walls gave a characteristic fragment, EtO2-P-Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-ManNAc(beta 1----4)GlcNAc (where EtO2 = 1,2-ethylenediol and Gro = glycerol). The results lead to the conclusion that in the cell walls of this organism, the ribitol teichoic acid chain is linked to peptidoglycan through a novel linkage unit, Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-(3/4)ManNAc-(beta 1----4)GlcNAc.

Synthesis of chorionic gonadotropin subunits in human choriocarcinoma clonal cell line JEG-3: carbohydrate differences in glycopeptides from free and combined alpha-subunits.

The glycoprotein hormone hCG and its free alpha-subunit are secreted by the clonal choriocarcinoma cell line JEG-3. Free hCG alpha has a larger apparent mol wt (22,000-24,000) than the combined hCG alpha (18,000-19,000) obtained by dissociation of the hCG secreted by these cells. Techniques developed for the specific isolation and purification of the free and combined hCG alpha forms and for the preparation of glycopeptides from these subunits have permitted detection of the incorporation of D-[3H]glucosamine [( 3H]GlcN) and L-[3H]fucose into both alpha-subunit forms. Relative to their [35S]methionine content, 2.3-fold more [3H]GlcN and 6-fold more L-[3H]fucose were incorporated into free hCG alpha than into combined hCG alpha. Analyses of [3H]GlcN glycopeptides prepared from free and combined hCG alpha indicate that the 22,000- to 24,000-dalton subunit form contained more [3H]GlcN and 27% more of the GlcN metabolite N-acetylneuraminic acid than the 18,000- to 19,000-dalton hCG alpha-subunit, than both hCG alpha forms contained two major N-linked oligosaccharide chains differing primarily in their NeuAc content, and that most of the [3H]GlcN was incorporated as GlcN or metabolites of GlcN other than N-acetylneuraminic acid. These studies provide direct chemical evidence of a higher content of carbohydrate in the larger free alpha-subunit form.

12] Preparation of glycopeptide and oligosaccharide probes for receptor studies

This chapter discusses the preparation of glycopeptides and oligosaccharide probes for receptor studies. The majority of studies directed at examining the specificity of mammalian lectins have relied on monosaccharides and glycoproteins as probes. Glycoproteins yield results that are difficult to interpret because they generally bear multiple oligosaccharides of differing structure. Polyvalency, because of the presence of multiple oligosaccharides on a single polypeptide, adds another complicating feature to specificity studies. It is possible to circumvent these difficulties by utilizing characterized glycopeptides and oligosaccharides to examine the specificity and other properties of these lectins. The chapter describes the general methods for the preparation of both glycopeptides and oligosaccharides that can be radiolabeled and some of their applications. 14 C and 3 H can be introduced into glycopeptides by acylation with 14 C or 3 H-labeled acetic anhydride; however, the products obtained have a relatively low specific activity and require scintillation counting for detection. The introduction of a p-hydroxyphenyl moiety onto the amino terminus of glycopeptides makes it possible to prepare 125 I-labeled derivatives of much higher specific activity.

Isolation of cell-surface glycopeptides from bovine cerebral cortex that inhibit cell growth and protein synthesis in normal but not in transformed cells.

Glycopeptides were isolated from the cell surfaces of bovine cerebral cortex that could inhibit increase in cell numbers in tissue culture and cellular protein synthesis. This cell growth inhibition apparently affected all cells exposed, could completely block cell division in a reversible manner and synchronized BHK-21 cell cultures. Polyoma-virus-transformed BHK-21 cells were completely insensitive to the inhibitor. Fractionation of the inhibitor on a Bio-Gel P-100 column revealed two peaks of biologically active material eluting at apparent molecular weights of 45 000 and 10 000 with A 1cm,280 1% 11.0. Affinity purification of the inhibitory fractions on a Ulex europaeus agglutination I lectin column resulted in retention of the inhibitory activity, suggesting the inhibitor material was a glycopeptide. Subsequent elution with 0.10 M-fucose resulted in a 244-fold increase in the specific biological activity over the starting material. Although purified from bovine brain, the material could inhibit baby-hamster kidney cell protein synthesis by 50% at a concentration of 5 x 10(6) molecules per target cell. Analysis by competitive radioimmunoassay or immunoadsorption indicated that the bovine inhibitor was structurally related to, although not necessarily identical with, a similar inhibitory glycopeptide preparation that we had previously isolated from mouse brain.

18] Preparation and fractionation of glycopeptides

Publisher Summary This chapter presents procedure for preparation and fractionation of glycopeptides. In preparation, samples containing membranous material, delipidation improve the yield of glycopeptides in the subsequent proteolytic step. Delipidation is also needed to remove the glycolipids from the sample. The double extraction procedure with chloroform-methanol 2:1 and 1:2 has been widely used. The more recently developed method has the advantage that even the highly glycosylated glycolipids (polyglycosyl ceramides H) become solubilized, and most glycoproteins remain insoluble. Purification of the total glycopeptide fraction is usually carried out by gel filtration using Sephadex G-25 or other similar column packings. For group fractionation of glycopeptides, they are first subjected to fractionation by affinity chromatography on concanavalin A-Sepharose. Biantennary N-glycosidic glycopeptides bind weakly to the lectin, and can therefore be eluted with a low concentration of the hapten sugar. High-mannose glycopeptides bind strongly to the column and need a high concentration of hapten sugar for elution. Complex N-glycosidic glycopeptides with more than two branches as well as O-glycosidic glycopeptides do not bind to the lectin. These two fractions are separated by gel filtration after cleavage of the O-glycosidic carbohydrate–peptide linkages by mild alkaline borohydride treatment.

Alkali-labile oligosaccharides from glycopeptides of human milk-fat-globule membrane

In view of its origin in the apical membrane of the mammary secretory cell (Patton & Keenan, 1975), the milk-fat-globule membrane is of interest as a relatively readily acessible source of mammalian epithelial membrane for structural and immunological studies. The bovine milk-fat-globule membrane, particularly, has been analysed, and a number of reports have described its glycoprotein components (Kobylka & Carraway, 1972: Anderson et al., 1974; Murray et al., 1979). which have been shown to carry relatively large amounts of the alkalilabile tetrasaccharide N-acetylneuraminyl-(2+3)-~-~-galactopyranosyl (1 -3) [ N acetylneuraminyl(2+6)1Nacetyl D galactosamine (Newman et al., 1976; Farrar & Harrison, 1978). This tetrasaccharide was originally described by Thomas & Winzler (1969) as a component of human erythrocyte membranes and has been also detected on membranes of rat brain (Finne, 1975), liver and kidney (Krusius & Finne, 1977), and on erythrocyte membranes from horse and sheep (Glockner et al., 1976). The human milk-fat-globule membrane is less readily available on a large scale and has been correspondingly less studied. Nevertheless, electrophoretic patterns of human milk-fatglobule-derived glycoproteins have been compared with those of their bovine counterparts and species differences were observed in the abilities of the glycoproteins to interact with certain lectins (Murray et al., 1979). Moreover, differences have been detected in the carbohydrate components of glycoproteins derived from human and bovine milk-fat-globule membranes (Glockner et al., 1976: Newman & Uhlenbruck, 1977) and also on the exposed surfaces of the globules themselves (Farrar et al., 1980). We now report the preparation and fractionation of glycopeptides proteolytically cleaved from the surface of intact human milk-fat globules and the release of relatively complex oligosaccharide complexes by treatment of the glycopeptides with alkaline borohydride. Freshly-expressed human breast milk (1.5 litres) was centrifuged and washed repeatedly in double-distilled water to give washed fat globules (50g), which were incubated with Pronase at 37OC for 1 h, cooled, centrifuged and dialysed to give a solution of sialoglycopeptides containing approx. 8 mg of hexose and 1.25 mg of sialic acid. The soluble sialoglycopeptides were fractionated by gel filtration on Sephadex G-50, when two major hexose/sialic acid peaks, peaks I and 11, were obtained, containing 20 and 80% repsectively of the total sialic acid applied to the column. Fractions corresponding to peak I1 were combined and applied to a column of Sephadex G-25. Elution with 0.1 M-acetic acid gave an excluded peak, fractions from which were combined so as to exclude a minor tail of hexose/sialic acid material. The pooled fractions were freeze-dried, treated with alkaline borohydride, freed from inorganic salts and rechromatographed on Sephadex G-25 as described above. Fractions eluted after the excluded peak were combined, freeze-dried and applied to a column of DEAE-Sephadex A-25. Elution with 0.05 M-pyridine acetate, followed by a linear gradient (0.05-0.65 M) of pyridine acetate, gave three hexose/ sialic acid peaks (HA, IIB and IIC), only the first (IIA) of which contained protein. Fractions corresponding to peak IIB, the larger of the protein-free peaks were combined, freeze-dried and chromatographed on preparative thin-layer plates of silica-gel G (development was with propan-2-ol/butan-2-ol/water), when two major bands, with R, 0.19 and 0.15 respectively, were obtained. The combined bands were eluted from the gel, freeze-dried and analysed for monosaccharide composition by using gas chromatography, when sialic acid, galactose, Nacetylglucosamine and N-acetylgalactosamine were found to be present in the molar ratio 6 : 7 : 4 : 2. Alkali-labile oligosaccharides released from mammalian membrane glycoproteins and fully characterized have largely been found to be structurally derived from the tetrasaccharide named above, which contains just sialic acid, galactose and N-acetylgalactosamine. Indications of more complex structures, containing glucosamine, have been obtained from bovine erythrocytes (Emerson & Kornfeld, 1976; DeWaard et al., 1976; Newman & Uhlenbruck, 1977), and it may be that such complexes, present also on human milk-fat-globule membranes, are more common than has been hitherto appreciated.

Isolation and characterisation of glycopeptides from digests of human tamm-horsfall glycoprotein

Glycopeptides were isolated from pronase digests of human Tamm-Horsfall glycoprotein and its asialo derivative. The carbohydrate moiety of the major glycopeptide preparation isolated from the former digests had an apparent molecular weight of 4300, and those of two glycopeptides isolated from the latter digests had molecular weights of 3600 and 2300. These data, together with the compositions of the glycopeptides, indicate that the Tamm-Horsfall glycoprotein has at least five asparagine residues substituted by complex carbohydrate moieties, three being of one type, relatively rich in galactose, and two containing more sialic acid but less galactose. A small amount of a mannose-rich glycopeptide was also recovered from the digests of the Tamm-Horsfall glycoprotein.

Almond glycopeptidase acting on aspartylglycosylamine linkages. Multiplicity and substrate specificity

The glycopeptide preparation that has been isolated from almond emulsin and acts on β-aspartylglycosylamine linkages in glycopeptides was separated into three active fractions by DEAE-cellulose column chromatography. The three discrete species of glycopeptidase (Groups A, B and C) have been purified 30-, 136- and 99-fold, respectively. The optimum pH value of Group A was 6.0 and those of Groups B and C, 5.0. Isoelectric points of Groups A, B and C were pH 7.7, 8.6 and 8.7, respectively. All three glycopeptidases hydrolyzed quantitatively glycopeptides with 3.11 amino acid residues prepared from stem bromelain, ovalbumin and ovotransferrin. Group C preferred glycopeptides with shorter peptide chains, whereas Groups A and B preferred those with longer chains. Glycopeptidase Group A also hydrolyzed intact glycoproteins such as stem bromelain, ovalbumin, Taka-amylase A and desialyted human transferrin.

Highly active amino-terminal sialoglycopenta-, hexa- and heptapeptides from human erythrocyte NM antigens.

Blood group Mand N-specific structures on human erythrocytes are carried by the hydrophilic :~-1 glycopeptides of the M and N glycoproteins [1 3]. Like the blood group ABH(O)-specific haptens [4, 5], those of the MN antigens are oligosaccharides as shown by mild acid hydrolysis [6], hemagglutination inhibition [2] and biosynthesis [7]. Most but not all Mand Nspecific groupings are located at the NH2terminus of the M and N antigens [2]. We describe here isolation and characterization of highly blood-group-active NH2terminal sialoglycopenta-, hexa-, and heptapeptides (SGPs). These novel SGPs were prepared by proteolysis with trypsin and pronase followed by column chromatography. From NN erythrocytes were obtained a sialoglyco(SG)-hexaand a SGheptapeptide, from MM red blood cells a SG-pentapeptide. All hydroxyamino acids of these three SGPs have glycosidically linked carbohydrate substituents. The Mand N-specific alkali-labile carbohydrate structures from Nand M-derived SGPs are different [2, 3] as is their amino acid composition [3, 8]. The M-derived SGP possesses, in addition to the tetrasaccharide, predominant in M and N glycoproteins, which was first described by Thomas and Winzler [9] (W.T.), one larger oligosaccharide chain. This chain, unless re-N-acetylated after the mild fl-elimination, appeared as two components on chromatographic separation. This large oligosaccharide amounted to ca. 35% of the total carbohydrate. Preparation of tryptic glycopeptides from whole erythrocytes and isolation of the c~-i SGPs was similar to our earlier procedure but was done on a larger scale. All serological and chemical .values given in the text and Tables represent arithmetic averages of at least three determinations; all carbohydrate analyses were carried out by two different standard analytical procedures [2, 6]. After rechromatography the c~-I SGP preparation moved as one component on polyacrylamide and thin-layer electrophoresis (TLE). The average yield of M-derived ~-1 SGP exceeded that of the ~-1 originating from N erythrocytes by 11% (wt.); in addition M-derived ~-1 SGP had 9% more NAN per unit weight (15 consecutive preparations of each c~-I N and e-1 M). Proteolysis of the ~-1 SGPs was with pronase (after its prior autodigestion to de,stroy possibly contaminating glycosidases). The digest was separated on two interconnected Sephadex G150 and G100 columns [2]. Five major fractions (I-V) were obtained, of which the fourth, because of its high activity of both Mas well as N-derived material and its relatively small molecular size, was studied in detail. Neither the Mnor the N-derived fractions IV were homogeneous by chromatography on Sephadex G-50 columns. Four and 5 elution peaks (SGP IV1_ 5) for Mand N-derived materials, respectively, were observed; their elution patterns were similar only for peak 1. SGP IV2M (peak 2) had highest M activity and had the NH2-terminal Ser; it had ca. 3.5% of ~-1 M SGP's total weight as well as NAN content (eight preparations average). In contrast, among the N-derived GP IV fractions, peak 1 had the highest N activity; it amounted to ca. 6% of e-1 SGP N by weight and had ca. 4% of its NAN (six preparations). The still impure SGP IV2M and IVIN were further purified by repeated DEAE-Sephadex A-25 ion exchange column chromatography using 0.I-1.0 M NH4HCO3, pH 7.8 gradient elution. SGP IV2M and SGP IV1N each yielded 5 subfractions, a-e. The most active fraction from SGP IV2M was c and from IV,N, e. Both amounted to 40 50% of the weight of the recovered materials.

Preparation of glycopeptides and oligosaccharides from thyroxine-binding globulin.

Over 99% of thyroxine (T4), the major form of thyroid hormone in plasma, is bound to the plasma glycoprotein thyroxine-binding globulin (TBG). The carbohydrate composition of TBG (14.6% by weight) consists of mannose, galactose, N-acetylglucosamine, and N-acetylneuraminic acid in the molar ratios of 11:9:16:10 per mol of glycoprotein. No fucose or N-acetylgalactosamine were detected. Amino acid analyses were performed. Glycopeptides, prepared by exhaustive pronase treatment of the glycoprotein, were separated by gel filtration and ion exchange chromatography. All glycopeptides contained the four sugars present in the native glycoprotein. One-fourth of the glycopeptide fraction was resolved into a discrete component, glycopeptide I. The remaining glycopeptides were a mixture termed glycopeptides II and III. Glycopeptides II and III were resolved into two discrete carbohydrate units, termed oligosaccharides A and B, by alkaline-borohydride treatment and DEAE-cellulose chromatography. We propose that TBG contains four oligosaccharide chains as calculated from the molecular weights of the glycopeptides and from compositional data assuming 1 asparagine residue/glycopeptide. The carbohydrate structures of the glycopeptides and relative affinities of TBG, glycopeptides and oligosaccharides for hepatocyte plasma membrane binding are presented in the accompanying paper (Zinn, A.B., Marshall, J.S., and Carlson, D.M. (1978) J. Biol. Chem. 253, 6768-6773.

Alkali-stable blood group A- and B-active poly(glycosyl)-peptides from human erythrocyte membrane

The study of the chemical nature of the ABH blood group antigens of the human erythrocyte membrane has been advanced in recent years by the isolation and characterization of several glycosphingolipids with blood group activity [l-3] . It has been reported that blood group ABH activity is present also in glycoproteins of the erythrocyte membrane [4-91. However, since these observations have been mainly based on serological inhibition tests, the presence of blood group-active glycolipids in the glycoprotein preparations cannot be excluded. Owing to the water solubility of the large-molecular size blood groupactive glycosphingolipids (the poly(glycosyl)ceramides), it has been suggested that these glycosphingolipids may have been regarded in some studies as glycoproteins [3]. The occurrence of protein-bound blood group ABH antigens in the erythrocyte membrane has therefore still been a matter of some controversy. In the present paper, we present direct chemical evidence for the occurrence of protein-bound blood group ABH antigens in the erythrocyte membrane. This was accomplished by the preparation of glycopeptides with pronase digestion from lipid-free membrane residues and the isolation of the blood group Aand B-active fractions using the a-galactosyland a-Nacetylgalactosaminyl-binding lectin [ 10,l l] of Bandeiraea simplicifolia (BS I lectin). The glycopeptides are composed of about 50-60 sugar residues/

Vitamin A-dependent fucose-glycopeptide from rat tracheal epithelium

Tracheas from normal and vitamin A-deficient rats were incubated in the presence of l-[ 3H]fucose and d-[ 14C]glucosamine to label epithelial and mesenchymal glycoproteins. The epithelium and the cartilage were separated by incubation with testicular hyraluronidase and processed separately for the preparation of glycopeptides. One major epithelial glycopeptide was doubly labeled. In vitamin A deficiency the amount of l-[ 3H]fucose was reduced to 33% that of the normal. The tracheal fucose-glycopeptide, in molar ratios, contained fucose 1.0, mannonse 1.0, galactose 0.4, hexasamine 4.7, and sialic acid 3.5. Sulfate was absent.

Convulsion-producing property of the dialyzable glycopeptide preparation from whole rat brain

A dialyzable preparation containing whole rat brain glycopeptides caused clonic-tonic convulsions when administered intraventricularly to cats. It was suggested that this effect is due to a glycopeptide recovered from a brain glycoprotein or glycoproteins.

Glycoproteins in brain tissue of the O-variant of GM2 gangliosidosis.

Abstract— The dialysableglycopeptide preparation recovered from the glycoproteins in cerebral gray matter of a case of the O‐variant form of GM2 gangliosidosis contained four fold more N‐acetylglucosamine and mannose than a similar preparation from normal gray matter. In the O‐variant form of GM2 gangliosidosis, the enzymes β‐N‐acetylhexosaminidases A and B are missing. A three‐ and four‐fold elevation, respectively, of N‐acetylglucosamine and mannose in the dialysable glycopeptide preparation from a case of Tay‐Sachs disease (B‐variant form of GM2 gangliosidosis) was noted. The B‐variant lacks hexosaminidase A but has ample supplies of hexosaminidase B. The brain level of glycosaminoglycans was not affected in the O‐ and B‐variant forms of GM2 gangliosidosis.

Glycopeptides from rat brain glycoproteins

The lipid-free protein residue of rat brain tissue was treated with papain to solubilize the heteropolysaccharide chains of the tissue glycoproteins. The glycopeptides were separated into non-dialyzable and dialyzable glycopeptide preparations. Each preparation was then sorted out into groups of glycopeptides by means of electrophoresis and gel filtration. The quantitatively predominant glycopeptides were the alkali-stable glycopeptides (Group A) which accounted for 64% of the glycopeptide carbohydrate recovered from rat brain. Most of the group A glycopeptides appeared in the non-dialyzable preparation. The molecular weight of the glycopeptides of Group A ranged from approximately 5200–3700. The largest glycopeptide molecule in this mixture possessed the highest electrophoretic mobility and contained one fucose, four N-acetylneuraminic acid (NANA), six N-acetylglucosamine, four galactose, and three mannose residues per molecule. The spectrum of glycopeptides isolated in this group showed a progressive decrease in NANA rsidues, NANA and galactose residues, and NANA, galactose, and N-acetylglucosamine residues which could be correlated with a progressive decline in molecular weight and electrophoretic mobility. Some of the glycopeptides in each fraction recovered from this group of glycopeptides contained sulfate ester groups. A second group of glycopeptides (Group C glycopeptides) accounted for 25% of the total glycoprotein carbohydrate recovered from rat brain. These were recoverd from the dialyzable glycopeptide preparation, and resolved into three fractions by column electrophoresis. These glycopeptides do not contain sulfate, are composed predominately of mannose and N-acetylglucosamine, and possess a molecular weight of approximately 3000. Several minor groups of glycopeptides were detected. Alkali-labile glycopeptides (Group B) appeared in the non-dialyzable glycopeptide preparation. The dialyzable glycopeptide preparation contained glycopeptides (Group E) which contained N-acetylgalactosamine and glucose. These had a molecular weight of approximately 2000. Group D glycopeptides recovered from the dialyzable glycopeptide preparation contained variable amounts of NANA, mannose, galactose, N-acetylglucosamine, and sulfate. These possessed a molecular weight of approximately 2900.

Phosvitin, a phosphoglycoprotein: Composition and partial structure of carbohydrate moiety

The composition and partial structure of the carbohydrate prosthetic group of phosvitin have been investigated, using a glycopeptide preparation obtained from the pronase digest of the parent protein. The composition of the carbohydrate unit, as expressed in nearest integers, is 3 moles of mannose, 3 of galactose, 5 of N -acetyl-glucosamine, and 2 of sialic acid per mole. The structure of the oligosaccharide moiety of the glycopeptide was studied by sequential enzymic hydrolysis using neuraminidase, β -galactosidase, β-N -acetylglucosaminidase, and α -mannosidase and sequential Smith degration. Two moles of sialic acid followed by 2.4 moles of galactose were released per mole of glycopeptide when it was treated sequentially with neuraminidase and β -galactosidase. On treatment of the intact glycopeptide with β -galactosidase, 1 mole of galactose was liberated, thus showing that the carbohydrate has a branched structure in which two sialic acids and one galactose are terminal residues. In the first cycle of the sequential Smith degradation, one N -acetylglucosamine residue was oxidized and removed, in addition to 2 moles of sialic acid and 3 moles of hexose. This is followed by oxidation of 2 N -acetylglucosamines (2nd cycle of Smith degradation) and one residue each of mannose and N -acetylglucosamine (3rd cycle of Smith degradation). From these pieces of evidence and those of the enzymic studies a tentative partial structure is proposed for the carbohydrate moiety of phosvitin.

Comparative studies of Lorenzini jelly from two species of elasmobranch: Part I. Preparation of glycopeptides

Glycopeptides from the Lorenzini jelly of two species of elasmobranch have been prepared and their sugar and amino acid components assayed. Preliminary structural studies are reported.