Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver.

2.50
Hdl Handle:
http://hdl.handle.net/10541/106581
Title:
Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver.
Authors:
Lyon, Malcolm; Gallagher, John T
Abstract:
Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.
Affiliation:
Cancer Research Campaign Department of Medical Oncology, Christie Hospital, Manchester, U.K.
Citation:
Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver. 1991, 273(Pt 2):415-22 Biochem. J.
Journal:
Biochemical Journal
Issue Date:
15-Jan-1991
URI:
http://hdl.handle.net/10541/106581
PubMed ID:
1846741
Type:
Article
Language:
en
ISSN:
0264-6021
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorLyon, Malcolmen
dc.contributor.authorGallagher, John Ten
dc.date.accessioned2010-06-21T14:03:57Z-
dc.date.available2010-06-21T14:03:57Z-
dc.date.issued1991-01-15-
dc.identifier.citationPurification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver. 1991, 273(Pt 2):415-22 Biochem. J.en
dc.identifier.issn0264-6021-
dc.identifier.pmid1846741-
dc.identifier.urihttp://hdl.handle.net/10541/106581-
dc.description.abstractHeparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.en
dc.language.isoenen
dc.subject.meshAnimals-
dc.subject.meshCentrifugation, Density Gradient-
dc.subject.meshChromatography, Gel-
dc.subject.meshChromatography, Ion Exchange-
dc.subject.meshElectrophoresis, Polyacrylamide Gel-
dc.subject.meshFemale-
dc.subject.meshGlycosylation-
dc.subject.meshGuanidine-
dc.subject.meshGuanidines-
dc.subject.meshHeparan Sulfate Proteoglycans-
dc.subject.meshHeparin Lyase-
dc.subject.meshHeparitin Sulfate-
dc.subject.meshLiver-
dc.subject.meshMale-
dc.subject.meshMesylates-
dc.subject.meshMolecular Weight-
dc.subject.meshPeptide Mapping-
dc.subject.meshPolysaccharide-Lyases-
dc.subject.meshProteochondroitin Sulfates-
dc.subject.meshRats-
dc.subject.meshRats, Inbred Strains-
dc.subject.meshSerine Endopeptidases-
dc.titlePurification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver.en
dc.typeArticleen
dc.contributor.departmentCancer Research Campaign Department of Medical Oncology, Christie Hospital, Manchester, U.K.en
dc.identifier.journalBiochemical Journalen

Related articles on PubMed

All Items in Christie are protected by copyright, with all rights reserved, unless otherwise indicated.