scholarly journals A sub-population of keratan sulphates derived from bovine articular cartilage is capped with α(2–6)-linked N-acetylneuraminic acid residues. Affinity chromatography using immobilized Sambucus nigra lectin and characterization using 1H n.m.r. spectroscopy

1992 ◽  
Vol 286 (1) ◽  
pp. 231-234 ◽  
Author(s):  
G H Tai ◽  
H G Morris ◽  
G M Brown ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Affinity Chromatography ◽  
Gel Permeation Chromatography ◽  
Similar Proportion ◽  
Lectin Affinity Chromatography ◽  
Sambucus Nigra ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Acetylneuraminic Acid ◽  
Sambucus Nigra Agglutinin ◽  
Femoral Head Cartilage

Alkaline borohydride-reduced keratan sulphate (KS) chains derived from bovine femoral head cartilage were fractionated by lectin affinity chromatography with Sambucus nigra agglutinin (SNA) into binding and non-binding populations. Analysis of the SNA-binding and non-binding KS chains using 600 MHz 1H n.m.r. spectroscopy showed that the former population contained alpha(2-6)-N-acetylneuraminic acid residues and the latter contained primarily alpha(2-3)-N-acetylneuraminic acid residues as chain terminators. Both populations contained a similar proportion of alpha(2-3)-N-acetylneuraminic acid residues within their protein-linkage regions, and similar sulphation and fucosylation levels. Analysis of these two fractions by gel-permeation chromatography (g.p.c.) on a TSK-30 XL column showed them to have the same size distributions. It was concluded from the n.m.r. spectra and g.p.c. data that the populations differed primarily in the mode of linkage of the chain-terminating sialic acids.

scholarly journals Structural and immunological studies of keratan sulphates from mature bovine articular cartilage

1989 ◽  
Vol 260 (1) ◽  
pp. 277-282 ◽  
Author(s):  
D J Thornton ◽  
H G Morris ◽  
G H Cockin ◽  
T N Huckerby ◽  
I A Nieduszynski ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Articular Cartilage ◽  
High Performance ◽  
Gel Permeation Chromatography ◽  
Spectroscopic Studies ◽  
Progressive Increase ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Femoral Head Cartilage

Two populations of alkaline-borohydride-reduced keratan sulphate (KS) chains were prepared from the two peptido-keratan sulphate trypsin fragments of proteoglycan aggregates isolated from bovine femoral head cartilage (6-year-old animals). Each population was separated by high-performance ion-exchange chromatography on a Pharmacia Mono-Q column into eight pools, Q1-Q8. These were analysed by gel permeation chromatography, radioimmunoassay with the monoclonal antibody MZ15, and 500 MHz 1H n.m.r. spectroscopy. Upon chromatography on Sephadex G-75 the Mono-Q fractions were shown to increase in hydrodynamic size progressively from Q1 to Q8 for both KS populations. For each population the strongest antigenic response with the anti-KS monoclonal antibody MZ15 was expressed by the two fractions of greatest size and charge density, Q7 and Q8. Proton n.m.r. spectroscopic studies on the two series of fractions demonstrated: (i) a progressive increase in the level of galactose sulphation from Q1 to Q8, (ii) the presence of approximately one alpha(1-3)-linked fucose residue per chain in every sample, and (iii) the presence of N-acetylneuraminic acids in three discrete environments, two alpha(2-3)- and one alpha(2-6)-linked in every sample. The results are discussed in terms of a possible heterogeneity in the carbohydrate-protein linkage region of keratan sulphates from bovine articular cartilage.

scholarly journals Fucose content of keratan sulphates from bovine articular cartilage

1991 ◽  
Vol 273 (2) ◽  
pp. 307-310 ◽  
Author(s):  
G H Tai ◽  
G M Brown ◽  
H G Morris ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Molecular Weight ◽  
Articular Cartilage ◽  
Molecular Size ◽  
Gel Permeation Chromatography ◽  
Repeat Sequence ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Gel Permeation ◽  
Fucose Content ◽  
Galactose Content

Alkaline-borohydride-reduced keratan sulphate chains were isolated from bovine articular cartilage (6-8-year-old animals). Nine keratan sulphate fractions of increasing molecular weight were prepared by gel-permeation chromatography on a calibrated column of TSK 30 XL. The samples were analysed for fucose and galactose contents (% by wt. of keratan sulphate) and fucose/galactose ratio. The fucose content increased with molecular size, but the galactose content remained constant. It was concluded that the alpha(1→3)-linked fucose [Thornton, Morris, Cockin, Huckerby, Nieduszynski, Carlstedt, Hardingham & Ratcliffe (1989) Biochem. J. 260, 277-282] was located within the poly-N-acetyl-lactosamine repeat sequence of articular-cartilage keratan sulphate.

scholarly journals Age-related changes in the structure of the keratan sulphate chains attached to fibromodulin isolated from articular cartilage

1998 ◽  
Vol 330 (2) ◽  
pp. 753-757 ◽  
Author(s):  
M. Robert LAUDER ◽  
N. Thomas HUCKERBY ◽  
A. Ian NIEDUSZYNSKI ◽  
H. K. Anna PLAAS
Keyword(s):  
Articular Cartilage ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
Acetylneuraminic Acid ◽  
Age Related ◽  
Keratanase Ii ◽  
Age Related Changes ◽  
Related Increase

Bovine articular cartilage fibromodulin has been isolated from animals aged 3 months to 8 years, and the attached keratan sulphate (KS) chains digested with keratanase II. The oligosaccharides generated have been reduced, examined by high-pH anion-exchange chromatography and their structures identified by comparison with standards. It has been shown that in fibromodulin from young articular cartilage, the KS chains do not possess either non-reducing terminal (α2-6)-linked N-acetylneuraminic acid or fucose (α1-3)-linked to sulphated N-acetylglucosamine residues. However, an age-related increase has been observed in the abundance of both (α2-6)-linked N-acetylneuraminic acid and (α1-3)-linked fucose, neither of which is found in KS isolated from non-articular cartilage, irrespective of the age of the source. Interestingly, the KS chain length remains constant as a function of age, which possibly relates to a role in collagen fibril assembly. In addition, no significant age-related changes were identified in levels of galactose sulphation.

scholarly journals A non-reducing terminal fragment from tracheal cartilage keratan sulphate chains contains α(2-3)-linked N-acetylneuraminic acid

1991 ◽  
Vol 278 (3) ◽  
pp. 779-785 ◽  
Author(s):  
J M Dickenson ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Anion Exchange ◽  
Chondroitin Sulphate ◽  
Tracheal Ring ◽  
Exchange Chromatography ◽  
Borohydride Reduction ◽  
Anion Exchange Column ◽  
Keratan Sulphate ◽  
Sialidase Activity ◽  
Acetylneuraminic Acid ◽  
Femoral Head Cartilage

Keratan sulphate chains were isolated from bovine tracheal ring cartilage (15-18-month-old animals) after papain digestion of the tissue followed by ethanol fractionation, chondroitinase ABC digestion and alkaline borohydride reduction. The keratan sulphate chains were further purified by anion-exchange chromatography on a Pharmacia Mono-Q column in order to remove any contaminating chondroitin sulphate and O-linked oligosaccharides. The chains were then treated with keratanase and the digest was subjected to alkaline borohydride reduction, producing oligosaccharides with galactitol at their reducing ends. The reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these, oligosaccharide (I), was shown by 500 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-3Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (I) The structure of this oligosaccharide shows that keratan sulphate chains from bovine tracheal ring cartilage may be terminated with N-acetylneuraminic acid linked alpha (2-3) to an unsulphated galactose. Keratan sulphate chains were also isolated from bovine femoral head cartilage (15-18-month-old animals) using an identical protocol, but with keratanase which was subsequently shown to have sialidase activity. This yielded oligosaccharide (II), the unsialyated version of (I): Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (II).

scholarly journals Skeletal keratan sulphate chains isolated from bovine intervertebral disc may terminate in α(2----6)-linked N-acetylneuraminic acid

1992 ◽  
Vol 282 (1) ◽  
pp. 267-271 ◽  
Author(s):  
J M Dickenson ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Intervertebral Disc ◽  
Anion Exchange ◽  
Gel Permeation Chromatography ◽  
Intervertebral Discs ◽  
Borohydride Reduction ◽  
Anion Exchange Column ◽  
Keratan Sulphate ◽  
Acetylneuraminic Acid ◽  
Sialidase Inhibitor ◽  
Gel Permeation

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (2-year-old animals) after digestion with chondroitin ABC lyase followed by digestion with diphenylcarbamoyl chloride-treated trypsin of A1D1 proteoglycans and gel-permeation chromatography on Sepharose CL-6B. The peptido-keratan sulphate fragments were subjected to alkaline borohydride reduction. The reduced chains were treated with keratanase in the presence of the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, and the digest was subjected to alkaline borohydride reduction. This produced oligosaccharides with galactitol at their reducing ends. This reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these was shown by 600 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-6Gal beta 1-4GlcNAc(6-SO4)beta 1-3Gal-ol The structure of this oligosaccharide shows that keratan sulphate chains from bovine intervertebral disc have non-reducing termini with N-acetylneuraminic acid linked alpha(2----6) as well as alpha(2----3) to an unsulphated galactose.

Activation energy and lectin affinity chromatography of gamma-glutamyltransferase as a marker for enzyme heterogeneity

1989 ◽  
Vol 22 (2) ◽  
pp. 115-119 ◽  
Author(s):  
Joris R. Delanghe ◽  
Marc L. De Buyzere ◽  
Ivan K. de Scheerder ◽  
Uwe Faust ◽  
Roger J. Wieme
Keyword(s):  
Activation Energy ◽  
Affinity Chromatography ◽  
Lectin Affinity Chromatography ◽  
Gamma Glutamyltransferase ◽  
Lectin Affinity ◽  
Enzyme Heterogeneity
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