A study of equilibrium binding of link protein to hyaluronate.
Link protein was extracted from bovine femoral-head cartilage, radiolabelled while in the proteoglycan-aggregate stage, and then purified by density-gradient centrifugation and gel chromatography. The purity of the preparation was assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and two species with approx. mol.wts. 45000 and 48000 were observed. Sedimentation-velocity experiments were performed in 0.5 M-guanidinium chloride/5 mM-phosphate, pH 7.4, and yielded an SO20, w of 4.75S. The proportion of link protein unable to interact with hyaluronate was determined by chromatography on Sepharose CL-4B. The binding of link protein to high-molecular-weight hyaluronate was studied by frontal-gel chromatography on Sepharose CL-4B in 0.5 M-guanidinium chloride/5 mM-phosphate/0.1% bovine serum albumin, pH 7.4. Experiments were performed at 10, 17 and 25 degrees C and the results were treated as described by Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672]. Dissociation constants of approx. (1-4) X 10(-8) M were obtained. The length of hyaluronate occupied per link-protein molecule was determined to be six to seven disaccharides.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D016326:Extracellular Matrix Proteins; D006820:Hyaluronic Acid / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011509:Proteoglycans; D000099249:Proteoglycan Link Protein
D016326:Extracellular Matrix Proteins; D006820:Hyaluronic Acid; D011506:Proteins; D011509:Proteoglycans; D000099249:Proteoglycan Link Protein