title: Proteoheparan sulfate from human skin fibroblasts. Isolation and structural characterization.
abstract: Fibroblasts in culture were incubated with [35S]sulfate/[3H]glucosamine or [35S]sulfate/[3H]leucine. Proteoglycans were isolated from the medium and a 4 M guanidinium chloride extract of the cell layer or from a trypsin digest of the cells and an extract of the cell residue. Proteoglycans were isolated by density gradient centrifugation, gel permeation, and ion exchange chromatography after digesting contaminating proteogalactosaminoglycans with chondroitinase ABC. Gel chromatography suggests that the cell-derived protoheparan sulfate had an Mr = 350,000 whereas the trypsin-released and the medium-derived counterparts both had an Mr = 140,000. Reduction and alkylation of the cell-derived proteoglycan gave rise to a component with Mr = 140,000, whilst the medium-derived form was not affected. Degradation of cell-associated proteoheparan sulfate by trypsin followed by papain or alkali suggest that the core protein consists of three types of regions, heparan sulfate-containing regions of Mr = 140,000, oligosaccharide-containing regions, and nonglycosylated peptide regions containing most of the [3H]leucine. The heparan sulfates of the cell- and medium-derived proteoglycans were similar in size distribution and charge density and with regard to the proportions and arrangements of various building blocks.
mesh_terms: D002478:Cells, Cultured; D002850:Chromatography, Gel; D005347:Fibroblasts / Q000378:metabolism; D006025:Glycosaminoglycans / Q000302:isolation & purification*; D006497:Heparan Sulfate / Q000096:biosynthesis / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D012867:Skin / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014316:Tritium; D014357:Trypsin
chemical_list: D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D010206:Papain; D010446:Peptide Fragments; D013462:Sulfur Radioisotopes; D014316:Tritium; D014357:Trypsin