scholarly journals Proteoglycans from pig aorta. Comparative study of their interactions with lipoproteins

1986 ◽  
Vol 235 (3) ◽  
pp. 823-831 ◽  
Author(s):  
J Wegrowski ◽  
M Moczar ◽  
L Robert
Keyword(s):  
Hyaluronic Acid ◽  
Ion Exchange ◽  
Chondroitin Sulphate ◽  
Gel Permeation Chromatography ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Hydrodynamic Size ◽  
Guanidinium Chloride ◽  
Protein Core ◽  
Gel Permeation

Different proteoglycans (PGs) were isolated from pig aorta for aggregation studies with hyaluronic acid and human low-density lipoproteins (LDL). Extraction of the intima-media with 4M-guanidinium chloride and digestion of the residue with collagenase solubilized 91% of aortic hexuronic acid content. From the guanidinium chloride extract two PGs were isolated by ion-exchange and gel-permeation chromatography: proteochondroitin sulphate (PGI) with a protein-core apparent Mr of 250 000 and proteodermatan-chondroitin sulphate (PGII) with a protein-core apparent Mr of 55 000. Only PGI forms high-Mr aggregates with hyaluronic acid. From the collagenase digest two other PGs were isolated: proteoheparan sulphate and proteochondroitin sulphate (PGIII and PGIV respectively). PGIV had a smaller hydrodynamic size than PGI. PGI and PGII formed insoluble complexes with human LDL in the presence of Ca2+. PGIII or PGIV did not form precipitates with the LDL. PGI and PGII, but neither PGIII nor PGIV, were bound to LDL-Sepharose. The main peaks of PGI and PGII were eluted from LDL-Sepharose with 60 mM- and 90 mM-NaCl respectively. The results indicate that aortic PGs have different interacting potentials with lipoproteins, depending on their Mr and their glycosaminoglycan composition.

scholarly journals Purification and renaturation of recombinant human interleukin-2

1987 ◽  
Vol 245 (1) ◽  
pp. 85-91 ◽  
Author(s):  
M P Weir ◽  
J Sparks
Keyword(s):  
Inclusion Bodies ◽  
Specific Activity ◽  
Interleukin 2 ◽  
Gel Permeation Chromatography ◽  
Reversed Phase ◽  
Reduced Form ◽  
Guanidinium Chloride ◽  
Denatured Protein ◽  
Additional Peak ◽  
Gel Permeation

Recombinant human interleukin-2 (IL-2) expressed as Escherichia coli was isolated as insoluble aggregates of protein (inclusion bodies) after cell breakage. IL-2 and contaminants were dissolved in 6 M-guanidinium chloride/10 mM-dithiothreitol, pH 8.5, and further purified in reduced and denatured form by gel-permeation chromatography in the same solvent. Renaturation was effected by dilution and autoxidation; IL-2 of native specific activity was isolated at over 95% purity by reversed-phase h.p.l.c.; an additional peak of reduced protein was also observed. Most losses of native IL-2 occurred on refolding, probably because of an aggregation process; concentrations around 1 microgram/ml were necessary to achieve 30% recovery. It was essential to maintain the denatured protein in reduced form before renaturation and autoxidation, which was most efficient at pH 8.5 with 1.5 microM-CuSO4. A procedure based on these observations has been used to prepare IL-2 on the 50 micrograms scale.

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.

Chitinases of Bacillus licheniformis B-6839: isolation and properties

1996 ◽  
Vol 42 (4) ◽  
pp. 307-315 ◽  
Author(s):  
Lesya A. Trachuk ◽  
Lyudmila P. Revina ◽  
Tatyana M. Shemyakina ◽  
Galina G. Chestukhina ◽  
Valentin M. Stepanov
Keyword(s):  
Ion Exchange ◽  
Bacillus Licheniformis ◽  
Gel Permeation Chromatography ◽  
The Other ◽  
Colloidal Chitin ◽  
Bacillus Circulans ◽  
Temperature Optimum ◽  
Chitinase A ◽  
Gel Permeation ◽  
Molecular Masses

Five chitinases were isolated from culture filtrates of Bacillus licheniformis B-6839 R and S variants by combination of hydrophobic, ion-exchange, and gel permeation chromatography. The enzymes had molecular masses of 66, 62, 53, 49, and 42 kDa. The chitinases revealed two activity optima against colloidal chitin at pH 4.5–5.5 and 9.0–9.5 and they were rather stable at pH 4.0–9.5. The temperature optimum of activity was 90 °C for the 62-kDa chitinase and 70 °C for the other enzymes. The 66-, 53-, and 42-kDa chitinases showed pronounced similarities in their N-terminal sequences and apparently belonged to the same group, which might be related to Bacillus circulans chitinase A1. The 49- and 62-kDa enzymes did not reveal structural similarities with other chitinases produced by the studied B. licheniformis strain. No relationship was found with the 89- and 76-kDa chitinases isolated earlier from B. licheniformis X-7u.Key words: Bacillus licheniformis, chitinase, multiplicity.

scholarly journals Bovine aortic chondroitin sulphate- and dermatan sulphate-containing proteoglycans Isolation, fractionation and chemical characterization

1981 ◽  
Vol 197 (2) ◽  
pp. 259-268 ◽  
Author(s):  
R Kapoor ◽  
C F Phelps ◽  
L Cöster ◽  
L A Fransson
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Proteinase Inhibitors ◽  
Chondroitin Sulphate ◽  
Periodate Oxidation ◽  
Chemical Characterization ◽  
Gel Chromatography ◽  
Carbohydrate Composition ◽  
Dermatan Sulphate ◽  
Guanidinium Chloride

1. Guanidinium chloride (4M) in the presence of proteinase inhibitors extracted 90% of bovine aorta galactosaminoglycans as proteoglycans that were subsequently purified by ion-exchange and gel chromatography. 2. Fractionation of the calcium salts of the purified proteoglycans with increasing concentration of ethanol yielded fractions PG-25 (28%), PG-35 (45%) and PG-50 (37%). 3. Fraction PG-50 contained proteochondroitin 6-sulphate, whereas fractions PG-25 and PG-35 were proteodermatan sulphates of greatly different carbohydrate composition; the molar proportions of L-iduronate-N-acetylgalactosamine 4-sulphate, D-glucuronate-N-acetyl-galactosamine 4-sulphate and D-glucuronate-N-acetylgalactosamine 6-sulphate were 75: 18 :7 in fraction PG-25 and 14 :46 :40 in fraction PG-35. 4. The presence of alternating or mixed sequences with L-iduronate- and D-glucuronate-containing repeating disaccharides was indicated by the formation of tetrasaccharides after chondroitinase AC digestion (single L-iduronate residues) and by the release of fragments containing four or five consecutive D-glucuronate-N-acetylgalactosamine repeats after periodate oxidation and alkaline elimination. 5. The amino acid compositions of fractions PG-25 and PG-35 were similar and markedly different from that of fraction PG-50, which also contained more side chains.

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