Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver.
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.
Lyon
M
M
Cancer Research Campaign Department of Medical Oncology, Christie Hospital, Manchester, U.K.
Gallagher
J T
JT
D000818:Animals; D002499:Centrifugation, Density Gradient; D011508:Chondroitin Sulfate Proteoglycans / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation / Q000187:drug effects; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D019812:Heparan Sulfate Proteoglycans; D019763:Heparin Lyase; D006497:Heparan Sulfate / Q000302:isolation & purification*; D008099:Liver / Q000737:chemistry* / Q000187:drug effects; D008297:Male; D008698:Mesylates / Q000494:pharmacology; D008970:Molecular Weight; D010449:Peptide Mapping; D011133:Polysaccharide-Lyases / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012697:Serine Endopeptidases / Q000494:pharmacology
D011508:Chondroitin Sulfate Proteoglycans; D019791:Guanidine; D006146:Guanidines; D019812:Heparan Sulfate Proteoglycans; D019763:Heparin Lyase; D006497:Heparan Sulfate; D008698:Mesylates; D011133:Polysaccharide-Lyases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; C012077:trifluoromethanesulfonic acid