Identification of core protein, an intermediate in proteoglycan biosynthesis in cultured chondrocytes from the Swarm...

After incubating cultured chondrocytes from the Swarm rat chondrosarcoma for 30 min with [3H]serine, a labeled macromolecule was found predominantly as Mr = approximately 370,000 species which was subsequently identified as a core protein precursor to cartilage proteoglycan from the following properties: (a) it was immunoprecipitated along with completed proteoglycan from cell extracts by an antiserum to the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid. Its immunoprecipitation could be inhibited completely by the addition of purified hyaluronic acid-binding region to the extracts, indicating the presence of common antigenic determinants with this region of the proteoglycan core protein. (b) the core protein precursor was able to interact with the hyaluronic acid and link protein in proteoglycan aggregates added as carrier to extracts to form mixed aggregates of high buoyant density in associative CsCl density gradients. Labeled core protein precursor and link protein were subsequently isolated from the mixed aggregates from the top of dissociative CsCl density gradients. (c) radioactivity in core protein precursor after a 30-min pulse of [3H]serine disappeared after inhibiting further protein synthesis with cycloheximide concurrent with the appearance of label in completed proteoglycan molecules.