Removal of glycosaminoglycans from cultures of human skin fibroblasts.
Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential medium containing [35S]sulphate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacter heparinum was added to the cell monolayers. This treatment removed most of the 35S-labelled glycosaminoglycans, with no appreciable removal of the 3H-labelled proteins or 3H-labelled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labelled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulphate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labelled glycosaminoglycans produced. Thus F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.
D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D005417:Flavobacterium / Q000201:enzymology; D006025:Glycosaminoglycans / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D019763:Heparin Lyase; D006801:Humans; D008297:Male; D011133:Polysaccharide-Lyases / Q000494:pharmacology; D012867:Skin / Q000187:drug effects / Q000378:metabolism*
D006025:Glycosaminoglycans; D006493:Heparin; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase