A FILTER-PAPER ASSAY FOR HYALURONIC-ACID SYNTHETASE - APPLICATION TO THE ENZYME FROM SWISS-3T3-FIBROBLASTS

An improved assay for hyaluronic acid (HA) synthetase is described tha t is suitable for rapid processing of large numbers of samples. High b ackground levels of unincorporated radioactivity are removed by passag e of the reaction through a Sephadex G-50 spin column. The labeled HA product is then precipitated onto glass fiber filters with cetylpyridi nium chloride. Apparent Km values for HA synthetase from Swiss 3T3 fib roblasts are 10.8 and 58.4 mu M for UDP-glucuronic acid and UDP-N-acet ylglucosamine, respectively. HA synthetase activity of quiescent cells is 4.5% of that found in actively growing cells and is stimulated in response to 10% calf serum. There is a greater than 10-fold increase i n HA synthetase activity when cells are harvested with hyaluronidase a s compared with trypsin.