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Biosynthesis of dermatan sulphate in cultured fibroblasts. Characterization of newly synthesized glycans from cells and microsomes.

Fibroblasts in culture were incubated with 35SO4 and [3H]glucosamine for periods ranging from 5 min to 24 h. 35S radioactivity was incorporated linearily into intracellular sulphated glycosaminoglycans for 10 min. At this time the rate of incorporation levelled off and radioactive glycans started to emerge in the pericellular pool. 20 min later radioactive glycans could be isolated from the medium. Incorporation of [3H]glucosamine into corresponding glycans was delayed by about 2-3 h. Dermatan sulphate isolated from the cells after half an hour of incubation contained much less iduronic acid than did chains obtained at a later times. The uronate composition of dermatan sulphate from the pericellular and medium fractions did not change appreciably during the incubation period. To obtain further information about the structure of newly synthesized glycosaminoglycans as well as intermediates thereof, microsomes were incubated with UDP-[14C]glucuronic acid, 3'-phosphoadenosine 5'-phospho-[35S]sulphate and UDP-N-acetylgalactosamine. Polymeric acceptors for the synthesis of hyaluronate, heparan sulphate and dermatan sulphate were present. Chondroitin, an intermediate in dermatan sulphate synthesis could also be demonstrated. The copolymeric structure of newly synthesized, microsomal, dermatan sulphate was similar to that of dermatan sulphate obtained after 0.5-1 h of synthesis in cultured cells.