Hyaluronate-protein complex of rous sarcoma virus-transformed chick embryo fibroblasts.
Involvement of covalently linked protein or peptide in the structure or synthesis of hyaluronate has not previously been convincingly demonstrated. We have developed conditions for double-labeling with [3H]leucine and [14C]acetate, then isolating and characterizing the cell-associated and secreted hyaluronate-protein complexes of Rous sarcoma virus-transformed chick embryo fibroblasts. The preparations were purified by Bio-Gel A-15m gel filtration and CsCl density gradient ultracentrifugation under dissociative conditions, followed by acid agarose gel electrophoresis in the presence of 0.1% Nonidet P-40. The purified hyaluronate preparations did not change their 3H:14C ratios after further sodium dodecyl sulfate or alkali treatment. The cell-derived hyaluronate-protein was resistant to pronase but susceptible to proteinase K in the presence of sodium dodecyl sulfate. After chondroitinase ABC digestion, the cell-derived 3H-labeled protein was separated from the 14C-labeled hyaluronate disaccharides, then shown to give a broad band corresponding to Mr approximately 12,000 on sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and to be susceptible to both pronase and proteinase K. The corresponding 3H-labeled peptide was prepared in the same manner from the medium hyaluronate and the [3H]leucine shown to be present in material smaller in amount and size than that from the cell. We propose from these and other published data that the cell-associated hyaluronate-protein may be bound to the cell surface and that the hyaluronate in the medium may be derived from it as a result of proteolytic scission.
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000378:metabolism*; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D002642:Chick Embryo; D005347:Fibroblasts / Q000378:metabolism; D006820:Hyaluronic Acid / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D011485:Protein Binding; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D014764:Viral Proteins; D006820:Hyaluronic Acid