Studies in immunochemistry

Morgan, W. T. J.; King, Hugh K.

doi:10.1042/bj0370640

Cited by 193 publications

(25 citation statements)

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“…The materials were examined for their capacity to inhibit specific haemagglutination as described by Morgan and King [25] using naturally occurring anti-A and anti-B agglutinins in normal human sera for measuring A and B activity. The H activity was measured using a rabbit immune anti-H serum kindly supplied by Professor W. M. Watkins, Lister Institute of Preventive Medicine, London S.W.l.…”

Section: Serological Examinationmentioning

confidence: 99%

“…A methylation study of the bronchial glycoprotein, neuraminidase-digested bronchial glycoprotein and mild acid-hydrolysed bronchial glycoprotein revealed that the N-acetylneuraminic acid was linked to C-3 of penultimate galactose units. Fucose occurs in three different linkages, approximately 50 % is linked to C-2 of penultimate galactose units, approximately 25 % is linked to C-4 of glucosamine residues already substituted at C-3 and the remainder is linked to C-3 of glucosamine residues already substituted at C-4.…”

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confidence: 99%

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Isolation and Characterisation of Glycoproteins from Sputum

Roberts

1974

European Journal of Biochemistry

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Sputum has been separated into a sol and a gel phase by ultracentrifugation and the components of these phases examined. A glycoprotein of high molecular weight as well as serum albumin, immunoglobulin A, sll acid glycoprotein and lactoferrin were detected in the sol phase by gel filtration and immunodiffusion studies. A glycoprotein of similar chemical composition was isolated from the gel phase by solubilisation in 6 M urea/O. 1 M NaCl followed by fractional precipitation with ethanol.The carbohydrate content of this glycoprotein, termed the bronchial glycoprotein, varied from 58 (w/w) to 69% (w/w) with sputum from different patients and was composed of fucose, galactose, galactosamine, glucosamine and N-acetylneuraminic acid. Serine, threonine and proline constituted 44% (w/w) of the amino acid residues of the bronchial glycoprotein and an alkaline borohydride cleavage study indicated that 0-glycosidic linkages exist between N-acetylgalactosamine and the hydroxyamino acids in the peptide core. Fractionation of the bronchial glycoprotein on DEAESephadex A-25 yielded fractions enriched in sulphate and sialic acid but separate fuco, sialo and sulpho glycoproteins were not obtained. Similarly fractionation of the oligosaccharides released by the action of alkaline borohydride yielded a complex mixture of oligosaccharides but sulphated oligosaccharides free of sialic acid were not obtained. Separation of the oligosaccharides into two fractions of different sizes showed that sulphate content was greatest in the larger oligosaccharides whereas sialic acid content was highest in the smaller oligosaccharides. A methylation study of the bronchial glycoprotein, neuraminidase-digested bronchial glycoprotein and mild acid-hydrolysed bronchial glycoprotein revealed that the N-acetylneuraminic acid was linked to C-3 of penultimate galactose units. Fucose occurs in three different linkages, approximately 50 % is linked to C-2 of penultimate galactose units, approximately 25 % is linked to C-4 of glucosamine residues already substituted at

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Section: Serological Examinationmentioning

confidence: 99%

mentioning

confidence: 99%

Isolation and Characterisation of Glycoproteins from Sputum

Roberts

1974

European Journal of Biochemistry

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“…AnaIytical density gradient sedimentation was performed in a Beckman model E analytical ultracentrifuge [5,6] Serological activity was determined by the capacity of the substances to inhibit in specific haemagglutination tests [17]. Fig.…”

Section: Sedimentationmentioning

confidence: 99%

Fractionation in a Density Gradient of some Glycoproteins from Human Ovarian Cyst Fluids

Dunstone

1969

European Journal of Biochemistry

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Equilibrium density gradient sedimentation in caesium chloride has been used as an eiTective method of separating the components of sulphite-reduced sparingly soluble glycoproteins from two human ovarian cyst fluids. An almost quantitative recovery of the carbohydrate and peptide constituents has been obtained. Some fractions had overall chemical compositions similar to those isolated previously by Dunstone and Morgan in 1965, while others contained much greater proportions of amino acids, especially of aspartic acid, glutamic acid and cystine.The results suggest that the sparingly soluble glycoproteins are large macromolecules containing serologically active carbohydrate chains, bound to specific peptide fragments which form an integral part of larger macromolecules, the insolubility of which is due to the presence of -S-S-bonds.The experiments emphasize the value of equilibrium density gradient sedimentation in the study of complex proteinpolysaccharide systems.Human blood group-specific glycoproteins are obtained from pseudomucinous ovarian cyst fluids by extraction of the freeze-dried fluids with 90--95O/, w/w phenol [l-31. I n most instances, water-soluble glycoproteins that have been well-characterized, and sparingly soluble glycoproteins about which little is known, are obtained. As a result of preliminary studies of the sparingly soluble gel-like substances Dunstone and Morgan [4], have suggested that these are large macromolecules composed of a t least two types of amino acid-containing structure associated through inter-molecular -S-S-bonds. One of these structures was identified, after reduction of the gel-like material, as a typical water-soluble blood group-specific glycoprotein. Although the other type of structure was not isolated as a discrete molecular species, the experiments suggested that it should contain a relatively high proportion of amino acids, especially of aspartic acid, glutamic acid and cystine. These conclusions were considered tentative, because the recoveries were not quantitative and because only a limited number of preparations were examined.Equilibrium density gradient sedimentation in caesium chloride is an effective method for the quantitative separation of proteinpolysaccharides with minimal degradation [5-81. The present paper describes the fractionation, by this method, of sulphitereduced, sparingly soluble glycoproteins from two human ovarian cyst fluids. Excellent recoveries of the carbohydrate and amino acid constituents were obtained. Some of the fractions had overall chemical compositions similar to those isolated previously The experiments emphasize the value of equilibrium density gradient sedimentation in the study of complex proteinpolysaccharide systems. MATERIAL AND METHODSOvarian Cyst Glycoproteins These were prepared as described by Dunstone and Morgan [a]. The sparingly soluble glycoproteins obtained from cyst fluids no.485 (groupA) and no. 531 (group B)' were reduced with sulphite at room temperature as follows [9] : 100 mg of the glycoprotein were suspende...

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“…Blood-group serological activities of all individual fucolipids were investigated according to Morgan and King [26] modified as follows. Mixed liposomes of individual glycolipid and sphingomyelin as auxiliary lipid [27] were prepared in NaCl/P, as described previously [5].…”

Section: Hemagglutination Inhibition Testsmentioning

confidence: 99%

Isolation and Purification of Lewis Blood-Group Active Glycosphingolipids from the Plasma of Human O Leb Individuals

Hanfland

1978

Eur J Biochem

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Eight different glycosphingolipids with four to six sugars per molecule have been isolated from human plasma of blood-group 0 Leh secretors. Purification was achieved by preparative highperformance thin-layer chromatography using a new silica gel. Homogeneity was checked by analytical high-performance thin-layer chromatography using four different solvent systems, by gas-liquid chromatographical analysis of the sugars as their alditol acetates revealing integral numbers of sugars and by the hemagglutination inhibition test. Two blood-group Lea-active ceramide pentasaccharides (glycolipids 4,5) and three blood-group Leh-active ceramide hexasaccharides (glycolipids 6, 7 and 8) were obtained, each revealing glucose, galactose, N-acetylglucosamine and fucose in molar ratios of 1 : 2: 1 : 1 (glycolipids 4,5) or 1 : 2: 1 : 2 (glycolipids 6, 7 and 8) respectively. 0.027 pg of glycolipids 6, 7 and 8 completely inhibited the agglutination of human 0 Le(a-b+) erythrocytes by 50 pl of 4 hemagglutinating units of caprine anti-Leh sera. Homogeneity of glycolipids 6, 7 and 8 was confirmed by the exclusion of contamination of the hypothetical Led antigen being a structural isomer of the Leh antigen: A crude neutral glycolipid fraction enriched in fucolipids from the plasma of 0 Le(a -b-) secretors showed a high-performance thin-layer chromatographical migration behaviour completely different from that of glycolipids 6, 7 and 8. Glycolipids 4 and 5 exhibited distinct Lea blood-group activities (0.4 pg of glycolipid 4 or 0.6 pg of glycolipid 5 per 50 pl of 4 hemagglutinating units of caprine anti-Lea serum), but were slightly contaminated by blood-group H activity or blood-group Leb activity (7 pg of glycolipid 4/100 p1 human anti-H serum and 1.7 pg of glycolipid 5/50 p1 caprine anti-Leh serum). 0.01 pg, 0.0006 pg or even only 0.0001 pg of glycolipid 6, 7 or 8 respectively are sufficient for incubation to convert 9 x lo7 0 Le(a-b-) erythrocytes into Leh-positive cells. This means in the case of glycolipid 8, that maximally 400 molecules per erythrocyte on the cell surface are sufficient to achieve an agglutinability by anti-Leb sera. In addition, two different globosides (glycolipids 1, 2) and a new ceramide tetrasaccharide (glycolipid 3 ) were isolated. The latter substance contains glucose, galactose and N-acetylglucosamine in molar ratios of 1 : 2: 1, but differs from the lacto-N-neotetraosylceramide by showing a distinctly slower high-performance thin-layer chromatographical migration behaviour .Ahhreviutions und Triviul Names. NaC1/Pi, phosphate-buffered saline (9 vol. 0.9% NaCl + 1 vol. isotonic phosphate buffer pH 7.4); R,-value, ratio between thin-layer chromatographical migration distance of individual glycolipid and thin-layer chromatographical migration distance of human erythrocyte membrane globoside; lacto-N-tetraose, Gal(B1-3)GlcNAc(fi1-3)Gal(P1-4)-Glc, lacto-N-neotetroase, Gal@-4)GlcNAc(p1-3)Gal(p1 -4)Glc; lacto-difucotetraose, Fuc(al-Z)Gal(pI -4)Glc(2tla)Fuc; for further details of lactotetraoses see [37]; globos...

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Studies in immunochemistry

Cited by 193 publications

References 9 publications

Isolation and Characterisation of Glycoproteins from Sputum

Isolation and Characterisation of Glycoproteins from Sputum

Fractionation in a Density Gradient of some Glycoproteins from Human Ovarian Cyst Fluids

Isolation and Purification of Lewis Blood-Group Active Glycosphingolipids from the Plasma of Human O Leb Individuals

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