Bird T A, Schwartz N B, Peterkofsky B
J Biol Chem. 1986 Aug 25;261(24):11166-72.
Our previous work showed that vitamin C deficiency caused about a 70-80% decrease in the incorporation of [35S]sulfate into proteoglycan of guinea pig costal cartilage, coordinately with a decrease in collagen synthesis (Bird, T. A., Spanheimer, R. G., and Peterkofsky, B. (1986) Arch. Biochem. Biophys. 246, 42-51). We examined the mechanism for decreased proteoglycan synthesis by labeling normal and scorbutic cartilage in vitro with radioactive precursors. Proteoglycan monomers from scorbutic tissue were of a slightly smaller average hydrodynamic size than normal but there was no difference in the size of the glycosaminoglycan chains isolated after papain digestion. The type of glycosaminoglycans synthesized and the degree of sulfation were unaffected as determined by chondroitinase ABC digestion and duel labeling with [35S]sulfate and [3H]glucosamine. Conversion of [3H]glucosamine to [3H]galactosamine also was unimpaired. There was about a 40% decrease in core protein synthesis, measured by [14C]serine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nevertheless, decreased incorporation of [35S]sulfate into scorbutic tissue persisted in the presence of p-nitrophenyl-beta-D-xyloside and cycloheximide, which indicated that the site of the scorbutic defect was beyond core protein synthesis and xylosylation. Galactosyltransferase activity in scorbutic cartilage decreased to about one-third the levels in control samples in parallel with the decreases in proteoglycan and collagen synthesis. Our results suggest that the step catalyzed by this enzyme activity, the addition of galactose to xylose prior to chondroitin sulfate chain elongation, is the major site of the scorbutic defect in proteoglycan synthesis. Decreased enzyme activity may be related to increased cortisol levels in scorbutic serum.
我们之前的研究表明，维生素C缺乏会导致豚鼠肋软骨蛋白聚糖中[35S]硫酸盐的掺入量减少约70 - 80%，同时胶原蛋白合成也减少（Bird, T. A., Spanheimer, R. G., and Peterkofsky, B. (1986) Arch. Biochem. Biophys. 246, 42 - 51）。我们通过用放射性前体体外标记正常和坏血病软骨来研究蛋白聚糖合成减少的机制。坏血病组织中的蛋白聚糖单体平均流体动力学尺寸略小于正常组织，但木瓜蛋白酶消化后分离出的糖胺聚糖链的大小没有差异。通过软骨素酶ABC消化以及[35S]硫酸盐和[3H]葡萄糖胺双重标记确定，合成的糖胺聚糖类型和硫酸化程度未受影响。[3H]葡萄糖胺向[3H]半乳糖胺的转化也未受损。通过[14C]丝氨酸掺入和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测量，核心蛋白合成减少了约40%。然而，在对硝基苯基 - β - D - 木糖苷和环己酰亚胺存在的情况下，坏血病组织中[35S]硫酸盐的掺入减少仍然存在，这表明坏血病缺陷的位点在核心蛋白合成和木糖基化之后。坏血病软骨中的半乳糖基转移酶活性降至对照样品水平的约三分之一，与蛋白聚糖和胶原蛋白合成的减少平行。我们的结果表明，该酶活性催化的步骤，即在硫酸软骨素链延长之前将半乳糖添加到木糖上，是蛋白聚糖合成中坏血病缺陷的主要位点。酶活性降低可能与坏血病血清中皮质醇水平升高有关。
