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The effect of beta-D-xylosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors.

ABSTRACT: Incorporation of [35S]]sulphate, [3H]glucose and [3H]serine into glycosaminoglycans and proteoglycans of embryonic-chicken sternum was measured in vitro in incubation medium containing 4-methylumbelliferyl beta-D-xyloside or p-nitrophenyl beta-D-xyloside at low concentrations, and in the absence of inhibitors of protein synthesis. Incorporation of sulphate was decreased by 80% in incubations in which 1mM-4-methylumbelliferyl beta-xyloside or 2.5 mM-p-nitrophenyl beta-xyloside was present; under these conditions, serum factors stimulated incorporation to only a small extent. When the concentration of the xyloside was decreased tenfold, incorporation of sulphate was inhibited by 60-70%, but when normal human serum or L-3,3',5-tri-iodothyronine or both were also added to the incubation medium, incorporation was markedly stimulated. Experiments in which [35S]sulphate and [3H]glucose were incorporated simultaneously, and enzymic analysis of glycosaminoglycans formed in such experiments, indicated that chondroitin sulphate formed in the presence of 0.1 mM-4-methylumbelliferyl beta-xyloside contained 30-40% less sulphate than did chondrotin sulphate synthesized in the absence of xylosides. Similar experiments, with [3H]serine instead of [3H]glucose, suggested also a 20-30% decrease in chain length of the chondroitin sulphate; this was confirmed by direct gel filtration of labelled glycosaminoglycans on a calibrated column. Incorporation of [3H]glucose or [3H]serine was stimulated by serum and tri-iodothyronine in parallel with incorporation of sulphate. The changes seen in the total chondroitin sulphate were mirrored in the major proteoglycan fraction, purified by isopycnic centrifugation of salt-extracted proteoglycans. The labelling pattern of chondroitin sulphate from this proteoglycan indicated that decreased sulphation of chondroitin sulphate was largely due to the inferior ability of short polysaccharide chains to accept sulphate, with some direct interference with transfer of sulphate to all chains. The results also suggested that the action of serum factors on synthesis of proteochondroitin sulphate is exercised at the level of either protein synthesis or transport to the sites of initiation of polysaccharide synthesis.

SUBMITTER: Gibson KD

PROVIDER: S-EPMC1164593 | biostudies-other | 1977 Feb

REPOSITORIES: biostudies-other

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Project description:1. Embryonic-chicken sterna, incubated in medium containing 0.1mm-4-methylumbelliferyl beta-d-xyloside (4-methylcoumarin 7-beta-d-xyloside), synthesize proteochondroitin sulphate that is significantly undersulphated and shorter than usual [Gibson, Segen & Audhya (1977) Biochem. J.162, 217-233]. 2. Neither the beta-d-galactoside nor the beta-d-glucuronide of 4-methylumbelliferone, nor 4-methylumbelliferone itself, produced the effects. The only metabolites of 4-methylumbelliferone that were detected in cartilages exposed to 4-methylumbelliferyl beta-d-xyloside were unchanged xyloside and chondroitin sulphate covalently attached to 4-methylumbelliferone. 3. Gel filtration of salt extracts of sterna incubated in medium containing the xyloside showed that there were two pools of chondroitin sulphate in the tissue. One pool was identified, on the basis of its elution pattern and the linear kinetics of incorporation of sulphate into it, as proteochondroitin sulphate. Incorporation into the other pool, whose properties suggested that it was methylumbelliferyl-chondroitin sulphate, indicated that it underwent partial turnover. The molecular weight of this chondroitin sulphate was about 19000, and it appeared to be about 70% sulphated. 4. When sterna were incubated in medium containing the xyloside, there was a very large incorporation of sulphate and glucose into glycosaminoglycans that were released into the incubation medium. This contrasts with incubations of sterna in the absence of the xyloside, in which less than 5% of the sulphate incorporated could be recovered from the medium. The glycosaminoglycan released into the medium was 4-methylumbelliferyl-chondroitin sulphate, whose average molecular weight was 7000-8000 and degree of sulphation more than 95%. 5. Incorporation of sulphate into proteochondroitin sulphate was stimulated more than 3-fold by addition of 20% (v/v) human serum and 10nm-l-3,3',5-tri-iodothyronine. Incorporation into methylumbelliferyl-chondroitin sulphate, in either the tissue or the medium, was not significantly altered. 6. The decrease in chain length and degree of sulphation of proteochondroitin sulphate is explained in terms of competition between peptide-linked primers and methylumbelliferone-containing primers at the intracellular sites of polysaccharidechain elongation and sulphation. The implications of the results for the mechanism of stimulation of proteoglycan synthesis by serum factors are discussed.