Evidence for rapid metabolic turnover of hyaluronate synthetase in Swarm rat chondrosarcoma chondrocytes

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Abstract
Synthesis of [3H]hyaluronate from [6-3H]glucosamine was investigated in cultures of Swarm rat chondrosarcoma chondrocytes treated with various concentrations (0.1 microM-0.1 mM) of cycloheximide for various times. Concentrations greater than 1 microM inhibited protein synthesis by greater than 90%. Hyaluronate synthesis was decreased, with a t1/2 for 50% inhibition of 80-120 min, depending on the concentration of cycloheximide present. Similar experiments using [1-3H]glucose as a precursor label gave similar results. Experiments using [6-3H]glucosamine as a precursor label and 0.18 mM-puromycin to inhibit protein synthesis inhibited hyaluronate synthesis (t1/2 = 82 min) with similar kinetics to cycloheximide-induced inhibition. Cultures incubated with 3.6 microM-cycloheximide for up to 9 h and supplemented with p-nitrophenyl beta-D-xyloside during the last 75 min of treatment showed increased synthesis of [3H,35S]chondroitin sulphate, demonstrating that UDP-hexose precursors for glycosaminoglycan synthesis are not rapidly depleted on blockage of protein synthesis. Rapid metabolic turnover of hyaluronate synthetase is the most likely cause for decreased hyaluronate synthesis in chondrocytes in which protein synthesis is inhibited.