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 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 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.

Improved separation and quantification of the "middle molecule" b4-2 in uremia.

1983 ◽  
Vol 29 (4) ◽  
pp. 703-707 ◽  
Author(s):  
P Faguer ◽  
N K Man ◽  
G Cueille ◽  
S Di Giulio ◽  
J L Funck-Brentano
Keyword(s):  
Peritoneal Dialysis ◽  
Healthy Subjects ◽  
Chromatographic Method ◽  
Removal Rate ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Middle Molecules ◽  
Gel Permeation ◽  
Uremic Patients ◽  
High Concentrations

Abstract The plasma of uremic patients usually contains high concentrations of the so-called middle molecules (molecular mass, 300 to 1500 Da), which exert various toxic effects. Among these numerous substances, only one, named peak b4-2, has been correlated with uremic neuropathy. We describe our improvement of a two-stage chromatographic method, gel permeation followed by anion-exchange chromatography (J. Chromatogr. 146: 55-65, 1978), for separation and quantification of b4-2 in body fluids. In analyzing more than 300 samples from 43 uremic patients and 12 healthy subjects, we found a linear correlation between peak area at 254 nm and b4-2 concentration in the range 0.8 to 32 mg/L. The coefficient of variation, including data acquired during seven changes of columns, was 9%. Analysis time (80 min) was shorter than required with other methods. Our results confirm previous data for urinary b4-2 excretion by healthy subjects and for b4-2 removal rate in uremic patients undergoing hemodialysis or hemofiltration. Patients treated with continuous ambulatory peritoneal dialysis have a higher b4-2 excretion than do healthy subjects, suggesting a higher production of this solute in uremic patients.

scholarly journals Osmoregulated Periplasmic Glucans ofErwinia chrysanthemi

2001 ◽  
Vol 183 (10) ◽  
pp. 3127-3133 ◽  
Author(s):  
Virginie Cogez ◽  
Philippe Talaga ◽  
Jerome Lemoine ◽  
Jean-Pierre Bohin
Keyword(s):  
High Performance ◽  
Large Fraction ◽  
Compositional Analysis ◽  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Variable Number ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Trichloracetic Acid ◽  
Laser Desorption Mass Spectrometry

ABSTRACT We report the initial characterization of the osmoregulated periplasmic glucans (OPGs) of Erwinia chrysanthemi. OPGs are intrinsic components of the bacterial envelope necessary to the pathogenicity of this phytopathogenic enterobacterium (F. Page, S. Altabe, N. Hugouvieux-Cotte-Pattat, J.-M. Lacroix, J. Robert-Baudouy and J.-P. Bohin, J. Bacteriol. 183:0000–0000, 2001 [companion in this issue]). OPGs were isolated by trichloracetic acid treatment and gel permeation chromatography. The synthesis of these compounds appeared to be osmoregulated, since lower amounts of OPGs were produced when bacteria were grown in media of higher osmolarities. However, a large fraction of these OPGs were recovered in the culture medium. Then, these compounds were characterized by compositional analysis, high-performance anion-exchange chromatography, matrix-assisted laser desorption mass spectrometry, and 1H and 13C nuclear magnetic resonance analyses. OPGs produced by E. chrysanthemi are very heterogeneous at the level of both backbone structure and substitution of these structures. The degree of polymerization of the glucose units ranges from 5 to 12. The structures are branched, with a linear backbone consisting of β-1,2-linked glucose units to which a variable number of branches, composed of one glucose residue, are attached by β-1,6 linkages in a random way. This glucan backbone may be substituted by O-acetyl andO-succinyl ester-linked residues.

Rapid analysis of bilirubin in neonatal serum. I. The binding of bilirubin to albumin.

1979 ◽  
Vol 25 (9) ◽  
pp. 1608-1612 ◽  
Author(s):  
K C Lu ◽  
K M Gooding ◽  
F E Regnier
Keyword(s):  
Serum Proteins ◽  
Binding Capacity ◽  
Gel Permeation Chromatography ◽  
Exchange Chromatography ◽  
Serum Samples ◽  
Binding Characteristics ◽  
Quenching Analysis ◽  
Gel Permeation ◽  
Bilirubin Binding

Abstract Evaulation of the severity of jaundice in the neonate may be determined by measuring the reserve binding capacity of serum proteins for free bilirubin. Determination of protein-bound bilirubin has been labor intensive, necessitating multiple runs on gel-permeation chromatography columns or, more recently, enzyme assays or fluorescence quenching analysis. We present a method for quantitation of free bilirubin and of bilirubin-binding capacity of serum by liquid chromatography. A gel-permeation column binds free bilirubin while allowing passage and quantitation of protein-bound bilirubin. Subsequent injection of a desorbing agent releases the adsorbed bilirubin from the column, permitting quantitation of free bilirubin. Bound and free serum bilirubin may be determined directly in less than 15 min using 10 microL of serum. The binding of bilirubin to neonatal serum is seen to be quite different from the binding to adult serum. Ion-exchange chromatography of adult and neonatal serum samples shows that their protein profiles are radically different. This difference probably accounts for the binding characteristics.

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.

Synthesis and characterization of 4-chloromethylstyrene polymers containing bulky organosilicon groups

e-Polymers ◽  
2004 ◽  
Vol 4 (1) ◽  
Author(s):  
Kazem Dindar Safa ◽  
Mirzaagha Babazadeh
Keyword(s):  
Differential Scanning Calorimetry ◽  
Glass Transition ◽  
Gel Permeation Chromatography ◽  
Synthesis And Characterization ◽  
Scanning Calorimetry ◽  
H Nmr ◽  
Gel Permeation ◽  
Covalently Linked ◽  
C Nmr

Abstract The homopolymer of 4-chloromethylstyrene and its copolymers with styrene (in 1:3 and 1:1 mole ratio) were synthesized by bulk and solution freeradical polymerisations, respectively, at 70±1°C using α,α'-azoisobutyronitrile as an initiator. Highly sterically hindered tris(trimethylsilyl)methyl (Tsi) substituents were then covalently linked to the obtained homopolymer and copolymers. The polymers were characterized by IR, 1H NMR and 13C NMR, differential scanning calorimetry (DSC) and gel permeation chromatography. DSC showed that incorporation of Tsi substituents in the side chains of homopolymer and copolymers increases the rigidity of the polymers and, subsequently, their glass transition temperature.

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