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Mouse mastocytoma cells were cultured in medium containing [3H]GlcN and concentrations of [35S]sulphate varying from 0.01 to 0.5 mM. Intracellular [35S]sulphate incorporation increased severalfold from the lowest concentrations, reaching a maximum at 0.1-0.2 mM, whereas incorporation of [3H]hexosamine remained constant at all sulphate concentrations. Proteo[3H]-chondroitin [35S]sulphate was isolated and incubated with chondroitin ABC lyase, yielding 35S-labelled and/or 3H-labelled delta Di-0S and delta Di-4S disaccharide products. The increasing percentage of delta Di-4S was consistent with the increasing sulphate incorporation at each higher [35S]sulphate concentration. Examination of proteochondroitin [35S]sulphate size by Sepharose CL-6B chromatography indicated a range consistent with various numbers of glycosaminoglycan chains on the protease-resistant serglycin core protein. Alkali-cleaved chondroitin [35S]sulphate products indicated similar size distributions at all sulphate concentrations with no indication of preferential sulphation being related to smaller or larger size. DEAE-cellulose chromatography of [3H]chondroitin [35S]sulphate glycosaminoglycans indicated a random undersulphation as [35S]sulphate concentration was lowered. Addition of 4-methylumbelliferyl beta-D-xyloside to the cultures resulted in a 2-2.5-fold stimulation of [3H]chondroitin [35S]sulphate synthesis with formation of beta-xyloside-[3H]chondroitin [35S]sulphate which was much smaller, as estimated by Sepharose CL-6B chromatography, than the decreased amount of [3H]chondroitin [35S]sulphate derived from proteo[3H]chondroitin [35S]sulphate. Much higher concentrations of sulphate were necessary to produce sulphation of the beta-xyloside-[3H]chondroitin comparable with that of proteo[3H]-chondroitin, as indicated by chondroitin ABC lyase products and DEAE-cellulose chromatography. The specific radioactivities of the [3H]GalN in the proteo[3H]chondroitin [35S]sulphate and beta-xyloside-[3H]chondroitin [35S]sulphate were calculated from the 3H and 35S c.p.m. of isolated dual-labelled delta Di-4S from each, and indicated that the presence of the beta-xyloside resulted in a dilution of the [3H]GlcN by endogenous GlcN that was 4 times higher than that of cultures lacking the beta-xyloside. The higher sulphate concentrations needed for sulphation of beta-xyloside-chondroitin suggests that the membrane-bound nature of the proteochondroitin acceptor in juxtaposition to a chondroitin sulphate-synthesizing enzyme complex effectively reduces the apparent Km for adenosine 3'-phosphate 5'-phosphosulphate.

Microsomal preparations from chondroitin 6-sulfate-producing chick embryo epiphyseal cartilage and from chondroitin 4-sulfate-producing mouse mastocytoma cells were incubated with varying concentrations of 3'-phosphoadenylylphospho[35S]sulfate and chondroitin hexasaccharide in the presence or absence of Triton X-100. [35S]Sulfate incorporation into hexasaccharide and into endogenous microsomal chondroitin 6-sulfate or endogenous microsomal chondroitin 4-sulfate was measured. With both microsomal systems, Triton X-100 increased the incorporation of [35S]sulfate into hexasaccharide but had much less effect on the incorporation into endogenous chondroitin sulfate. Higher concentrations of hexasaccharide inhibited the incorporation of [35S]sulfate into endogenous chondroitin sulfate. The apparent Km for 3'-phosphoadenylylphosphosulfate for both the 6-sulfotransferase and 4-sulfotransferase with hexasaccharide and with endogenous chondroitin sulfate in the presence or absence of Triton X-100 were all similar. However, the apparent Km for hexasaccharide was lower in the presence of Triton X-100 for both the microsomal sulfotransferases. This is consistent with solubilization of sulfotransferases, and indicates that hexasaccharide access to these enzymes had been limited in the particulate system. Examination of 35S-oligosaccharide products formed with each system demonstrated good 6-sulfation or 4-sulfation of penultimate GalNAc at the non-reducing end of the chondroitin hexasaccharide. However, no 6-sulfation of terminal GalNAc at the non-reducing end of a chondroitin pentasaccharide derived from hexasaccharide was observed, and there was only minimal 4-sulfation of this terminal GalNAc. Concurrent addition of GalNAc to hexasaccharide resulting in heptasaccharide did not appear to promote significant 6-sulfation or 4-sulfation of newly added non-reducing terminal GalNAc.