scholarly journals Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue

1990 ◽  
Vol 269 (1) ◽  
pp. 55-59 ◽  
Author(s):  
J M Dickenson ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Ester Group ◽  
Intervertebral Discs ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Linkage Region ◽  
Gel Permeation ◽  
Electron Micro Probe Analysis

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].

scholarly journals Xyloglucan endotransglycosylase: evidence for the existence of a relatively stable glycosyl–enzyme intermediate

1998 ◽  
Vol 330 (3) ◽  
pp. 1475-1480 ◽  
Author(s):  
Zdena SULOVÁ ◽  
Miriam TAKÁČOVÁ ◽  
M. Nancy STEELE ◽  
C. Stephen FRY ◽  
Vladimír FARKAŠ
Keyword(s):  
Complex Formation ◽  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glycosidic Bond ◽  
Xyloglucan Endotransglycosylase ◽  
Anomeric Configuration ◽  
Gel Permeation ◽  
Covalently Linked ◽  
Enzyme Intermediate

Xyloglucan endotransglycosylases (XETs) catalyse the breakdown of xyloglucan molecules predominantly by transglycosylation. In this process, fragments of cleaved polysaccharide are preferentially transferred to other xyloglucan molecules or their oligosaccharide subunits, with overall retention of the anomeric configuration of the glycosidic bond. In accordance with the theory, we propose that the cleavage and re-formation of the glycosidic bond in xyloglucan involves the formation of a glycosyl-enzyme intermediate which decomposes by transfer of the glycosyl moiety to a suitable carbohydrate acceptor. XETs from nasturtium seed cotyledons, mung bean hypocotyls and cauliflower florets interacted with xyloglucan to form complexes of high Mr as judged by gel-permeation chromatography. The nasturtium enzyme also showed evidence of XET-xyloglucan complex-formation according to anion-exchange chromatography and adsorption of the complex to filter paper on the basis of affinity of its xyloglucan moiety for cellulose. The XET-xyloglucan complex was stable in water, 6 M urea and acidic and alkaline buffers (pH 2.5-9.5), but readily decomposed by transferring its glycosyl moiety to xyloglucan-derived oligosaccharides or by incubation with the strong nucleophile imidazole at pH 3.8-9.6. These results strongly support the assumption that XET forms a relatively stable covalently linked glycosyl-enzyme intermediate.

scholarly journals Separation of a series of chromophores and fluorophores present in elastin

1975 ◽  
Vol 147 (2) ◽  
pp. 215-219 ◽  
Author(s):  
D P Thornhill
Keyword(s):  
Optical Properties ◽  
Ion Exchange ◽  
Fluorescence Spectra ◽  
Cation Exchanger ◽  
Gel Permeation Chromatography ◽  
Protein Molecule ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Intact Protein ◽  
Gel Permeation

Purified elastin was hydrolysed with HCl and manipulated under conditions that minimized oxidation. Gel-permeation chromatography on polyacrylamide gel and ion-exchange chromatography on dextran cation-exchanger each resulted in the separation of a series of yellow fluorescent fractions. These hitherto unreported ampholytes have fluorescence spectra that approximate to that of the intact protein, and account for its characteristic optical properties. Since the coloured fluorophores are confined to enzyme-resistant regions of the protein molecule they appear to have important structural implications.

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.

Quantitative fractionation of casein mixtures by fast protein liquid chromatography

1987 ◽  
Vol 54 (3) ◽  
pp. 369-376 ◽  
Author(s):  
D. Thomas Davies ◽  
Andrew J. R. Law
Keyword(s):  
Liquid Chromatography ◽  
Ion Exchange ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fast Protein Liquid Chromatography ◽  
Good Resolution ◽  
Gel Permeation

SummaryAlkylation of whole casein samples by reaction with cysteamine and cystamine in a bis-tris-propane–urea buffer (pH 7·0) followed by fast protein liquid chromatography (FPLC) at 20°C on a Mono Q HR5/5 column in the same buffer and using a NaCl gradient led to good resolution of the whole casein into fractions representing (i) γ2- plus γ3-caseins, (ii) κ-caseins, (iii) β-casein, (iv) αs2-caseins and (v) αsl-caseins, together with small amounts of unidentified materials. Quantitatively the FPLC values agreed well with those for αs1-, β-, αs2- and γ2- plus γ3-caseins obtained by ion-exchange chromatography on DEAE cellulose, Whatman DE52 and with those for º-caseins obtained by gel-permeation chromatography on Sephadex G–150.

Frequent Occurrence of HIV-Inhibitory Sulphated Polysaccharides in Marine Invertebrates

1993 ◽  
Vol 4 (3) ◽  
pp. 167-172 ◽  
Author(s):  
J. A. Beutler ◽  
T. C. McKee ◽  
R. W. Fuller ◽  
M. Tischler ◽  
J. H. Cardellina ◽  
...  
Keyword(s):  
Marine Invertebrates ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Aqueous Extracts ◽  
Sulphated Polysaccharides ◽  
Anionic Polysaccharides ◽  
Gel Permeation ◽  
Anti Hiv ◽  
C Nmr

Aqueous extracts of many marine invertebrates have exhibited some activity in the National Cancer Institute's primary screen for anti-HIV cytopathicity. Using a variety of techniques, including gel permeation, size exclusion and ion exchange chromatography, toluidine blue metachromicity, 13C-NMR spectroscopy and combustion analyses, we have determined that this activity is largely due to sulphated polysaccharides. Because of the wide occurrence of this class of compounds in these organisms we sought a method for the rapid dereplication of sulphated polysaccharides. It was critical that the method selected for dereplication allow differentiation of anionic polysaccharides from other AIDS-antiviral chemotypes. After evaluating a variety of methods, we found that the most efficient strategy appeared to be precipitation of the polysaccharide fraction from aqueous ethanolic solutions of the crude aqueous extracts.

Isolation of a spermatozoal immobilization factor from Staphylococcus aureus filtrates

2009 ◽  
Vol 55 (7) ◽  
pp. 874-878 ◽  
Author(s):  
Vijay Prabha ◽  
Tanushree Gupta ◽  
Siftjit Kaur ◽  
Navchetan Kaur ◽  
Sushila Kala ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gel Permeation Chromatography ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Human Spermatozoa ◽  
Infertile Woman ◽  
Ammonium Sulfate Precipitation ◽  
Tail Region ◽  
Sperm Immobilization

Staphylococcus aureus isolated from the cervix of an infertile woman was found to cause complete immobilization of human spermatozoa in vitro. Only the cell culture and cell-free supernatant showed immobilization activity, indicating that the sperm immobilization factor might be released extracellularly by the organism because no activity was observed with the washed cells. Heat treatment of the supernatant at 60 °C for 10 min waived its immobilizing activity, indicating that the active component may be a protein. The bioactive molecule from the supernatant was purified to homogeneity by ammonium sulfate precipitation, gel permeation chromatography, and ion exchange chromatography. Sperm immobilization factor (SIF) was found to be an ~20 kDa protein. SIF at a concentration of 10 µg/mL was required to cause 100% immobilization of human spermatozoa after 30 min of incubation at 37 °C, whereas a concentration of 150 µg/mL caused immediate immobilization, and a concentration of 200 µg/mL resulted in instant loss of viability of human spermatozoa, observed by eosin–nigrosin staining. Scanning electron microscopy showed that the treatment of human spermatozoa with SIF caused multiple defects in the head, midpiece, neck, and tail region of human spermatozoa.

scholarly journals Hyaluronic acid in cartilage and proteoglycan aggregation

1974 ◽  
Vol 139 (3) ◽  
pp. 565-581 ◽  
Author(s):  
Timothy E. Hardingham ◽  
Helen Muir
Keyword(s):  
Hyaluronic Acid ◽  
Buoyant Density ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Sulphate Content ◽  
Lower Protein ◽  
Proteoglycan Aggregates

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.

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 Structural analysis of a new series of oligosaccharide-alditols released by reductive β-elimination from oviducal mucins of Rana utricularia

1998 ◽  
Vol 330 (1) ◽  
pp. 469-478 ◽  
Author(s):  
Willy MORELLE ◽  
Gérard STRECKER
Keyword(s):  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amphibian Species ◽  
Structural Investigations ◽  
Jelly Coat ◽  
Specific Material ◽  
Species Specific ◽  
Carbohydrate Chains ◽  
Rana Utricularia

Egg jelly coats from Rana utricularia are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they pass along the different oviducal portions. In this study, carbohydrate chains of the jelly coat surrounding the eggs of R. utricularia were released by alkali/borohydride treatment. Fractionation of O-linked oligosaccharide-alditols was achieved by a combination of chromatographic techniques comprising anion-exchange chromatography, gel-permeation chromatography and HPLC on a silica column bonded with aminopropyl groups. Structural characterization was performed by one- and two-dimensional 1H-NMR spectroscopy in combination with matrix-assisted laser-desorption ionization-time of flight MS and methylation analysis. Ten oligosaccharide structures possessing a core consisting of Galβ(1 → 3)GalNAc-ol with or without branching through a GlcNAc residue linked β(1 → 6) to the GalNAc residue (core type 2 or core type 1 respectively) are described. The most representative carbohydrate sequences are: GlcNAc(β1-3)[Fuc(α1-4)]GlcNAc, GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc(β1-3)GlcNAc and Gal(β1-3)GlcNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc. The carbohydrate chains isolated from R. utricularia are quite different from those found in other amphibian species, in which the presence of species-specific material has been characterized. Since the jellies surrounding amphibian eggs are involved in egg-sperm interactions, these structural investigations can provide biochemical support for investigation of the fertilization process.

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