Isolation and Characterization of Mouse Articular Cartilage Proteoglycans Using Preformed CsCl Density Gradients

A technique has been developed for the rapid isolation of proteoglycans from small amounts of tissue extracts using the Beckman Airfuge. Aliquots (50 p1) of proteoglycan samples are layered on preformed CsCl density gradients and centrifuged at -135,000 X g for 4 h. Under both associative and dissociative conditions, proteoglycan sediments to the lower third of the tube (e.g. on a preformed associative gradient, 93% of the uronic acid in a bovine nasal cartilage extract accumulates in this bottom fraction). Gel chromatography indicates predominately aggregate in the bottom third of the associative gradient, and gel electrophoresis of ‘261-labeled fractions reveals that the proteoglycan is well separated from other proteins in the extract. The uronic acid distribution through a dissociative fractionation is similar to that obtained by conventional equilibrium density gradient centrifugation. Gel chromatography of the monomer fraction indicates the absence of hyaluronic acid and a broad included peak typical of proteoglycan monomer from bovine nasal cartilage. Enzymatic assay for hyaluronic acid shows it to be predominately in the top third of the tube. Both gel electrophoresis and quantitation by radioimmunoassay show the link proteins in the top fraction. This rapid fractionation of small sample volumes in the Airfuge is used in conjunction with additional modifications of extraction procedures to isolate and characterize mouse articular cartilage proteoglycan. The modifications, meant to minimize proteolytic degradation of the proteoglycans, include short extraction and dialysis times and maintenance of subzero temperatures. The ‘2gII-labeled mouse proteoglycan of the aggregate fraction is excluded from Sepharose CL-2B, and the K., of the ‘2gI-labeled mouse monomer is 0.46. Reacting this ‘261-labeled aggregate with a link-specific antiserum reveals only the smaller link protein 2 in mouse articular cartilage aggregate.