title: Cartilage link proteins. Biochemical and immunochemical studies of isolation and heterogeneity.
abstract: Native and clostripain-derived link proteins from proteoglycan aggregates were separated by gel chromatography in 4 M guanidine HCl from low-buoyant-density proteoglycan and proteoglycan hyaluronic acid-binding region after extraction from adult bovine nasal cartilage and the Swarm rat chondrosarcoma. Separations were monitored with Laurell immunoelectrophoresis using precipitating antibodies to link protein, hyaluronic acid-binding region, and low-buoyant-density proteoglycan. These immunoanalyses, with sodium dodecyl sulfate-polyacrylamide gel analyses, revealed the usefulness of this combined approach when assessing the purities and identities of these molecules. Using these isolated link proteins we provide data suggesting the presence of two immunologically detectable forms of link proteins of the same molecular weight. On immunoelectrophoresis both rat and bovine link proteins of a single molecular weight each produced two precipitin reactions with antibody to link protein. They were of similar intensity for native molecules but of different intensity for clostripain-isolated link protein. Isoelectric focusing in 6 M urea revealed that the 48,000 and 44,000 molecular weight link proteins of bovine nasal cartilage together produce a complex pattern of many bands. Link proteins of a single molecular size produced a much simpler, predominantly five-banded focusing pattern. Immunoelectrophoresis of the electrofocused clostripain rat link protein (42,000) revealed that the three major central bands each produced double precipitin reactions. Mixing of native and clostripain-derived link protein in 4 M guanidine HCl followed by dialysis to 6 M urea prior to isoelectric focusing did not change the focusing position of the individual bands. This suggests that the heterogeneity of focusing forms was due to the existence of different isoforms. The double precipitin reactions may be due to the existence of two different conformations expressing different epitopes of the kind reported previously by Thonar et al. (Thonar, E. J.-M. A., Kimura, J. H., Hascall, V. C., and Poole, A. R. (1982) J. Biol. Chem. 257, 14173-14180).
mesh_terms: D000818:Animals; D002356:Cartilage / Q000032:analysis*; D002417:Cattle; D002813:Chondrosarcoma / Q000032:analysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D011506:Proteins / Q000302:isolation & purification*; D011509:Proteoglycans / Q000302:isolation & purification*; D051381:Rats; D013045:Species Specificity; D000099249:Proteoglycan Link Protein
chemical_list: D016326:Extracellular Matrix Proteins; D046911:Macromolecular Substances; D011506:Proteins; D011509:Proteoglycans; D000099249:Proteoglycan Link Protein