Isolation and characterization of the heparan sulfate proteoglycan of the bovine glomerular basement membrane.
A proteoglycan, accounting for about 75% of the total hexuronic acid of the bovine glomerular basement membrane, was solubilized by 4 M guanidine HCl extraction and purified by filtration on Sepharose CL-6B. This glycoconjugate was found to have an apparent molecular weight of 200,000 and to consist of peptide and carbohydrate in a ratio of 70 to 30. The amino acid composition of the proteoglycan was notable for its high content of half-cystine (61/1000 total amino acid residues). Alkaline NaB3H4 treatment of the proteoglycan released heparan sulfate chains terminating in [3H]xylitol with an Mr approximately 14,000 (hexuronic acid/xylitol = 30:1). Four such glycosaminoglycan units were calculated to be present in each proteoglycan molecule, and on the basis of previous studies (Parthasarathy, N., and Spiro, R. G. (1981) J. Biol. Chem. 256, 507-513), these appear to be clustered in a very limited segment of the polypeptide. Nitrous acid degradation of the NaB3H4-reduced chains yielded radiolabeled oligosaccharides derived from the xylitol end; the relatively large size of these fragments (average Mr approximately 6,000) indicated an uneven distribution of N-sulfate with a preferential location of these groups in the peripheral regions of the glycosaminoglycans. The occurrence in the proteoglycan of sugars such as galactosamine, sialic acid, and mannose, which are not constituents of heparan sulfate, suggested that other O-linked as well as N-linked carbohydrates occurred in the molecule. Evidence for small serine (threonine)-bound units was provided by alkaline NaB3H4 treatment of the proteoglycan which converted 85% of the galactosamine to 3H-labeled galactosaminitol present in Bio-Gel P-2 included oligosaccharides (14 mol/mol proteoglycan).
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001485:Basement Membrane / Q000032:analysis*; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D011508:Chondroitin Sulfate Proteoglycans / Q000302:isolation & purification*; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D006025:Glycosaminoglycans / Q000302:isolation & purification*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000302:isolation & purification*; D007678:Kidney Glomerulus / Q000032:analysis*; D011509:Proteoglycans / Q000302:isolation & purification*
D000596:Amino Acids; D002241:Carbohydrates; D011508:Chondroitin Sulfate Proteoglycans; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans