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A K Allen

Publications and source records attributed to A K Allen.

At least 55 records · Page 3Linked to original sources
PubMed1985

Comparative compositions of cell surface glycoconjugates isolated from Trypanosoma cruzi epimastigotes.

Cell surface glycoconjugates of epimastigotes of Trypanosoma cruzi have been isolated and analyzed to give their amino acid and carbohydrate compositions. Those which have been investigated are a complex of three closely associated glycoproteins, GP24, GP31, GP37, and a lipopeptidophosphoglycan. The GP24-GP31-GP37 complex has an unusual amino acid composition with very low levels of hydrophobic amino acids, it contains 56% (w/w) carbohydrate, with mannose, galactose and glucosamine (presumably N-acetyl) being present in approximately equal quantities. The lipopeptidophosphoglycan also has low levels of hydrophobic amino acids and contains equal levels of mannose and galactose together with lesser amounts of (N-acetyl) glucosamine. The glycoconjugates are contrasted and compared with two other previously characterised cell surface glycoproteins (GP25 and GP72) from T. cruzi.

Amino Acids↗
PubMed1984

A comparison of tomato (Lycopersicon esculentum) lectin with its deglycosylated derivative.

A deglycosylated derivative of tomato (Lycopersicon esculentum) lectin was prepared with the use of trifluoromethanesulphonic acid. Its properties were generally similar to those of the native lectin, but differences were evident in terms of relative agglutinating activity towards sheep, (untreated) human and trypsin-treated human erythrocytes. The two forms of tomato lectin were used in conjunction with a battery of specific antisera to investigate structural relatedness among solanaceous lectins. Immunological cross-reactivity between tomato, potato and Datura lectins depends on the integrity of the glycosylated region of those lectins; that between Datura lectin and other seed lectins, however, has a separate structural basis.

Cross Reactions↗
PubMed1983

Studies on the structure of a phosphoglycoprotein from the parasitic protozoan Trypanosoma cruzi.

A glycoprotein (GP72) has been isolated from Trypanosoma cruzi and found to contain 41% protein, 49% carbohydrate and 10% phosphate. All phosphate was covalently attached to the carbohydrate which contained the following sugars: ribose, xylose, fucose, galactose, mannose, glucose and glucosamine. The carbohydrate side chains were linked to protein by fucose, xylose and N-acetylglucosamine; 50% of the total N-acetylglucosamine was involved in glycoprotein linkages. Two classes of carbohydrate side chains were detected. One class comprised 15% of the total carbohydrate and contained glucosamine, mannose and galactose; some of these chains were phosphorylated. The other class comprised 85% of the total carbohydrate and contained xylose, ribose, fucose, galactose, mannose, glucosamine and phosphate; these chains were antigenic and reacted with a monoclonal antibody with specificity for the whole glycoprotein.

Acetylglucosamine↗
PubMed1983

The properties of potato (Solanum tuberosum) lectin after deglycosylation by trifluoromethanesulphonic acid.

Potato (Solanum tuberosum) lectin, which is a very highly glycosylated glycoprotein, has been completely deglycosylated by use of the trifluoromethanesulphonic acid reagent described by Edge, Faltnek, Hof, Reichert & Weber [(1981) Analyt. Biochem. 118, 131-137]. This shows that both hydroxyproline-arabinofuranoside and serine-galactopyranoside linkages are hydrolysed. The deglycosylated lectin is still functional and cross-reacts with one component of an anti-(potato lectin) antiserum.

Amino Acids↗
PubMed1983

Purification and preliminary biochemical characterisation of the human and rat forms of the central nervous system-specific molecule, F3-87-8.

The F3-87-8 monoclonal antibody recognises a phylogenetically conserved antigenic determinant found exclusively in the mammalian CNS. We used this monoclonal antibody as the major purification step for obtaining pure. F3-87-8-bearing molecules from rat and human brains for biochemical analysis. In both rat and man, the F3-87-8 molecule is a heavily glycosylated protein, consisting of 47.6 and 47.0% carbohydrate by weight, respectively. In both species, it occurs as a doublet on polyacrylamide gel electrophoresis in sodium dodecyl sulphate, the Mr of the human form being 130,000 and 100,000. In the rat, the Mr of the doublet is slightly but consistently lower than in man, and the higher-Mr band is more pronounced. The amino acid composition of the rat and human forms is virtually identical, with a high content of serine and threonine. Significant differences are seen in carbohydrate composition, the rat form containing more sialic acid and neutral sugar and less hexosamine than the human form. beta-Elimination studies, in conjunction with carbohydrate analysis, suggest the presence of approximately 40 O-linked and 10-15 N-linked oligosaccharides per polypeptide chain of 500 amino acids, the N-linked chains predominantly of the high-mannose type. This makes it likely that the molecule adopts an extended rather than a coiled conformation on the membrane.

Amino Acids↗
PubMed1982

The detection of phosphonolipids in the protozoan Trypanosoma cruzi.

2-Aminoethylphosphonate was detected in the acid hydrolysates of the phosphonolipids and the lipopeptidophosphoglycan of Trypanosoma cruzi, the causative agent of Chagas' disease. This finding represents the first evidence of phosphonolipids in a zooflagellate. By comparison, no phosphonolipids were detected in Trypanosama brucei, indicating that phosphonolipids are not a ubiquitous feature of the Order Kinetoplastidia.

Animals↗
PubMed1982

The production and properties of an antiserum to potato (Solanum tuberosum) lectin.

Precipitation of potato (Solanum tuberosum) lectin by antisera was not affected by treatments that abolish lectin activity. An antiserum precipitated glycosylated derivatives of the lectin but not a deglycosylated peptide. The haemagglutination inhibition titre of this antiserum was not affected by removing anti-glycopeptide antibodies. This evidence suggests that the antiserum contains two populations of antibodies, specific for different domains of the lectin.

Animals↗
PubMed1982

Structural studies of the carbohydrate moieties of lectins from potato (Solanum tuberosum) tubers and thorn-apple (Datura stramonium) seeds.

1. Methylation analysis of potato (Solanum tuberosum) lectin and thorn-apple (Datura stramonium) lectin confirmed previous conclusions that both glycoproteins contained high proportions of l-arabinofuranosides and lesser amounts of d-galactopyranosides. The arabinofuranosides are present in both lectins as short unbranched chains containing 1-->2- and 1-->3-linkages, which are known to be linked to hydroxyproline. Galactopyranosides are present as monosaccharides, which are known to be attached to serine, in potato lectin and as both the monosaccharide and the 1-->3-linked disaccharide in Datura lectin. 2. Alkaline digestion of potato lectin and subsequent separation of the components by gel filtration led to the isolation of four fractions corresponding to the mono-, di-, tri- and tetra-arabinosides of hydroxyproline. The latter two fractions accounted for over 70% of the total hydroxyproline. 3. Methylation analysis was used to show that the triarabinoside contained only 1-->2-linkages between sugars, but that the tetra-arabinoside contained both 1-->2- and 1-->3-linkages. Direct-insertion mass spectrometry of these compounds using electron impact and chemical ionization, in a comparison with other known structural patterns, was used to determine the sequences of the sugars, which were Araf1-->2Araf1-->2Araf1-->Hyp and Araf1-->3Araf1-->2Araf1-->2Araf 1-->Hyp. 4. On the basis of optical rotation it had previously been suggested [Allen, Desai, Neuberger & Creeth (1978) Biochem. J.171, 665-674] that all the arabinose of potato lectin was present as the beta-l-furanoside. However, measurement of the optical rotations of the hydroxyprolyl arabinosides showed that whereas the diarabinoside had a molar rotation ([m]) value close to that predicted, the triarabinoside was more dextrorotatory and the tetra-arabinoside was less dextrorotatory than expected. Possible explanations for these findings are that, although the di- and tri-arabinosides contain exclusively beta-arabinofuranosides, in the tri-arabinoside, interactions between pentose units lead to an enhanced positive rotation. The tetra-arabinoside, however, is proposed to contain a single alpha-arabinofuranoside residue, which is responsible for the lower than expected positive rotation. The observed rotation of the tetra-arabinoside was found to be close to the theoretical value predicted on that basis. Furthermore, the action of a specific alpha-arabinofuranosidase on the tetrasaccharide was to remove a single arabinose residue, presumably the terminal non-reducing sugar, and to produce a product that was indistinguishable on electrophoresis from the triarabinoside. Changes in rotation were compatible with this assumption. 5. It is concluded that the structures of the hydroxyprolyl tri- and tetra-arabinosides of potato lectin are: betaAraf1-->2betaAraf1-->2betaAraf1-->Hyp and alphaAraf1-->3betaAraf1-->2betaAraf 1-->2betaAraf1-->Hyp. These are identical with compounds that have been isolated from the insoluble hydroxyproline-rich glycoproteins of plant cell walls.

Amino Acids↗
PubMed1981

Studies on the chemical modification and potato (Solanum tuberosum) lectin and its effect on haemagglutinating activity.

1. Modification of potato (Solanum tuberosum) lectin with acetic anhydride blocked 5.1 amino and 2.7 tyrosyl groups per molecule of lectin and decreased the haemagglutinating activity of the lectin. De-O-acetylation regenerated 2.0 of the tyrosyl groups and resulted in a recovery of activity. 2. Modification with citraconic anhydride or cyclohexane-1,2-dione did not greatly affect activity, although modification of amino and arginyl groups could be demonstrated. 3. Treatment with tetranitromethane nitrated 3.7 tyrosine residues per molecule of lectin with concomitant loss of activity. The presence of 0.1m-NN'N''-triacetylchitotriose (a potent inhibitor of the lectin) in the reaction medium protected all the tyrosyl residues from nitration and the lectin was fully active. 4. Modification of tryptophyl groups with 2-hydroxy-5-nitrobenzyl bromide and 2,3-dioxoindoline-5-sulphonic acid modified 0.9 and 2.6 residues per molecule of lectin respectively with a loss of activity in each case. Reaction of potato lectin with 2,3-dioxoindoline-5-sulphonic acid in the presence of inhibitor protected 2.4 residues of tryptophan from the reagent. Loss of haemagglutination activity was prevented under these conditions. 5. Reaction of carboxy groups, activated with carbodi-imide, with alpha-aminobutyric acid methyl ester led to the incorporation of 5.3 residues of the ester per molecule of lectin. Presence of inhibitor in this case, although protecting activity, did not prevent modification of carboxy groups; in fact an increase in the number of modified residues was seen. This effect could be imitated by performing the reaction in 8m-urea. In both cases the number of carboxy groups modified was close to the total number of free carboxy groups as determined by the method of Hoare & Koshland [(1967) J. Biol. Chem.242, 2447-2453]. Guanidination of lysine residues after carboxy-group modification gave less homoarginine than did the unmodified lectin under the same conditions, suggesting the formation of intramolecular cross-links during carbodi-imide activation. 6. It is suggested from the results presented that amino, arginyl, methionyl, histidyl and carboxyl groups are not involved in the activity of the lectin and that tyrosyl and tryptophyl groups are very closely involved. These findings are similar to those reported for other proteins that bind N-acetylglucosamine oligomers and also fit the general trend in other lectins.

Amino Acids↗
PubMed1981

Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen.

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

Amino Acids↗
PubMed1981

Some properties of the lectin from Datura stramonium (thorn-apple) and the nature of its glycoprotein linkages.

The lectin from Datura stramonium (thorn-apple; Solanaceae) has been purified by affinity chromatography and shown to be a glycoprotein containing about 40% (w/w) of carbohydrate. The most abundant amino acids are hydroxyproline, cystine, glycine and serine. Results obtained by gel filtration in 6m-guanidinium chloride on Sepharose 4B suggest that it has a subunit mol.wt. of about 30000 and that it probably associates into dimers. The lectin is inhibited specifically by chitin oligosaccharides and bacterial-cell-wall oligosaccharides, but only weakly by N-acetylglucosamine. Glycopeptides from soya-bean (Glycine max) lectin and fetuin are also strong inhibitors of Datura lectin, indicating that it interacts with internal N-acetylglucosamine residues. Its specificity is similar to, but not identical with, that of potato (Solanum tuberosum) lectin. After prolonged proteolytic digestion of reduced and S-carboxymethylated or S-aminoethylated derivatives of the lectin, glycopeptides of mol.wt. of about 18000 were isolated. The glycopeptides contained all the carbohydrate and hydroxyproline of the original glycoprotein, and lesser amounts of serine, S-carboxymethylcysteine and other amino acids. The arabinose residues of the glycoprotein are present as beta-l-arabinofuranosides linked to the polypeptide chain through the hydroxyproline residues, and can be removed by mild acid treatment; the ratio of arabinose to hydroxyproline is 3.4:1. Some of the serine residues of the polypeptide chain are substituted with one or two alpha-galactopyranoside residues, most of which can be removed by the action of alpha-galactosidase. The galactose residues are more easily removed from the acid-treated glycopeptide (from which arabinose has been removed) than from the complete glycopeptide, indicating a steric hindrance of the galactosidase action by the adjacent chains of arabinosides. There is a slow release of galactose residues by a process of beta-elimination in 0.5m-NaOH (pH13.7) from the complete glycopeptide, and a fairly rapid release of galactose by this process from the acid-treated glycopeptide, which lacks arabinose. This is probably due to the inhibitory effect of the negative charge on the adjacent arabinofuranoside residues. The similarities and differences between the lectins from Datura and potato are discussed, as are their structural resemblance to glycopeptides that have been isolated from plant cell walls.

Amino Acids↗
PubMed1981

Cell surface antigens of Trypanosoma cruzi: use of monoclonal antibodies to identify and isolate an epimastigote specific glycoprotein.

A monoclonal antibody was produced by cell fusion from mice immunized with Trypanosoma cruzi epimastigotes. The antibody was epimastigote specific but not strain specific; the antibody bound to Y, Peru and Tulahuén epimastigotes but did not bind to Y amastigotes or trypomastigotes. The antigen recognised by the monoclonal antibody was a 72 000 molecular weight cell surface glycoprotein which represented only 0.04% of the whole cell protein. Analysis of the glycoprotein purified by antibody affinity chromatography revealed a carbohydrate content of 52% by weight which was composed of glucosamine, mannose, galactose, glucose and three different pentoses: fucose, xylose and ribose. Immunisation with the purified glycoprotein did not protect mice from a lethal infection of T. cruzi.

Animals↗
PubMed1979

A lectin from the exudate of the fruit of the vegetable marrow (Cucurbita pepo) that has a specificity for beta-1,4-linked N-acetylglucosamine oligosaccharides.

Lectins are present in the exudate (presumably from the phloem) of the fruits of three species of the Cucurbitaceae, namely vegetable marrow (Cucurbita pepo), melon (Cucumis melo) and cucumber (Cucumis sativus). They are all strongly inhibited in their activities by chitin oligosaccharides, but only weakly by N-acetylglucosamine. Glycopeptides from soya-bean agglutinin and fetuin are also strong inhibitors of Cucurbita pepo lectin, indicating that it interacts with internal N-acetylglucosamine residues. The lectin from Cucurbita pepo fruit was purified by affinity chromatography by using chitin oligosaccharides covalently attached to Sepharose. The lectin is not a glycoprotein, and it consists of a single polypeptide chain of about 20,000 mol.wt. It is a major protein (18% of the total) of the phloem exudate and it is postulated that it may have an anti-parasitic function.

Acetylglucosamine↗
PubMed1978

Properties of potato lectin and the nature of its glycoprotein linkages.

1. Potato lectin is a glycoprotein that contains about 47% (by weight) l-arabinose, 3% d-galactose and 11% hydroxyproline. It has a monomeric molecular weight of about 50000 and probably exists as a monomer-dimer system in aqueous solution, with the monomer predominating. It has a very high viscosity, which would indicate either that the molecule is very expanded or that it is an elongated ellipsoid. 2. After prolonged proteolytic digestion of a reduced and carboxymethylated derivative of the lectin, a glycopeptide was isolated (of mol.wt. 32000-34000) that included all the carbohydrate and hydroxyproline of the original glycoprotein but less than 30% of the total original amino acid residues. 3. The arabinose of the glycoprotein is present exclusively as the beta-arabinofuranoside and this includes those residues that are directly linked to the hydroxyproline residues of the polypeptide chain. All the arabinose of the glycoprotein is linked to the polypeptide chain through the hydroxyproline residues; the ratio of arabinose to hydroxyproline is 3.4:1. Although alpha-arabinofuranosides are known to be present in arabinans and arabinogalactans, the natural occurrence of beta-arabinofuranosides has not previously been reported. 4. Nine or ten serine residues of the polypeptide chain are substituted with single alpha-galactopyranoside residues that can be removed by the action of alpha-galactosidase from coffee beans but not by a beta-galactosidase. This is the first report of an alpha-galactoside linkage to serine. The effect of alpha-galactosidase is much greater on a glycopeptide from which the arabinose has been already removed, which indicates a steric hindrance of the galactosidase action by adjacent chains of arabinosides. 5. In 0.5m-NaOH (pH13.7), galactose residues were removed from the serine residues of the glycopeptide by a process of beta-elimination. This reaction took place very slowly in the intact glycopeptide but much more rapidly when the arabinofuranoside residues had been removed. This inhibitory effect of the arabinofuranoside residues on the beta-elimination reaction is likely to be due to a negative charge on the hydroxy groups of the adjacent arabinofuranoside residues, which would be ionized at this high pH value. 6. It is suggested that potato lectin may be representative of a class of soluble plant glycoproteins that would include precursors of the cell-wall glycoprotein extensin. If this is the case, extensin should also contain beta-l-arabinofuranosides linked to hydroxyproline and alpha-d-galactopyranosides linked to serine residues of the polypeptide chain.

Amino Acids↗
PubMed1978

Vicia faba lectin.

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Chromatography, Affinity↗