Effects of cyclo heximide on the biosynthesis and secretion of proteo glycans by chondrocytes in culture

Mitchell, D.; Hardingham, T.

Biochemical Journal 196(2): 521-530


ISSN/ISBN: 0264-6021
Accession: 005297830

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Proteoglycans synthesized by rat chondrosarcoma cells in culture are secreted into the culture medium through a pericellular matrix. The appearance of [35S]sulfate in secreted proteoglycan after a 5 min pulse was rapid (half-time, t1/2 < 10 min), but that of [3H]serine into proteoglycan measured after a 15 min pulse was much slower (t1/2 = 120 min). The incorporation of [3H]serine into secreted protein was immediately inhibited by 1 mM cycloheximide, but the incorporation of [35S]sulfate into proteoglycans was only inhibited gradually (t1/2 = 79 min), suggesting the presence of a large intracellular pool of proteoglycan that did not carry sulfated glycosaminoglycans. Cultures were pulsed with [3H]serine and [35S]sulfate and chased for up to 6 h in the presence of 1 mM cycloheximide. Analysis showed that cycloheximide-chased cells secreted less than 50% of the [3H]serine in proteoglycan of control cultures and the rate of incorporation into secreted proteoglycan was decreased (from t1/2 = 120 min to t1/2 80 min). Under these conditions cycloheximide interfered with the flow of proteoglycan protein core along the route of intracellular synthesis leading to secretion and inhibiting further protein-core synthesis. Apparently the newly synthesized protein core of proteoglycan passes through an intracellular pool for about 70-90 min before the chondroitin sulfate chains are synthesized on it, and it is then rapidly secreted from the cell. Proteoglycan produced by cultures incubated in the presence of cycloheximide and labeled with [35S]sulfate showed an increase with time of the average proteoglycan size and the length of the constituent chondroitin sulfate chain. The proportion of synthesized proteoglycans able to form stable aggregates did not alter.