McQuillan D J, Handley C J, Robinson H C, Ng K, Tzaicos C, Brooks P R, Lowther D A
Biochem J. 1984 Dec 15;224(3):977-88. doi: 10.1042/bj2240977.
The effect of cycloheximide on chondroitin sulphate biosynthesis was studied in bovine articular cartilage maintained in culture. Addition of 0.4 mM-cycloheximide to the culture medium was followed, over the next 4h, by a first-order decrease in the rate of incorporation of [35S]sulphate into glycosaminoglycan (half-life, t 1/2 = 32 min), which is consistent with the depletion of a pool of proteoglycan core protein. Addition of 1.0 mM-benzyl beta-D-xyloside increased the rate of incorporation of [35S]sulphate and [3H]acetate into glycosaminoglycan, but this elevated rate was also diminished by cycloheximide. It was concluded that cycloheximide exerted two effects on the tissue; not only did it inhibit the synthesis of the core protein, but it also lowered the tissue's capacity for chondroitin sulphate chain synthesis. Similar results were obtained with chick chondrocytes grown in high-density cultures. Although the exact mechanism of this secondary effect of cycloheximide is not known, it was shown that there was no detectable change in cellular ATP concentration or in the amount of three glycosyltransferases (galactosyltransferase-I, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II) involved in chondroitin sulphate chain synthesis. The sizes of the glycosaminoglycan chains formed in the presence of cycloheximide were larger than those formed in control cultures, whereas those synthesized in the presence of benzyl beta-D-xyloside were consistently smaller, irrespective of the presence of cycloheximide. These results suggest that beta-D-xylosides must be used with caution to study chondroitin sulphate biosynthesis as an event entirely independent of proteoglycan core-protein synthesis, and they also indicate a possible involvement of the core protein in the activation of the enzymes of chondroitin sulphate synthesis.
在体外培养的牛关节软骨中研究了放线菌酮对硫酸软骨素生物合成的影响。向培养基中添加0.4 mM放线菌酮后，在接下来的4小时内，[35S]硫酸盐掺入糖胺聚糖的速率呈一级下降（半衰期t1/2 = 32分钟），这与蛋白聚糖核心蛋白池的耗尽一致。添加1.0 mM苄基β-D-木糖苷可提高[35S]硫酸盐和[3H]乙酸盐掺入糖胺聚糖的速率，但该升高的速率也被放线菌酮降低。得出的结论是，放线菌酮对该组织有两种作用；它不仅抑制核心蛋白的合成，还降低了组织合成硫酸软骨素链的能力。在高密度培养的鸡软骨细胞中也获得了类似的结果。尽管放线菌酮这种次要作用的确切机制尚不清楚，但结果表明，细胞ATP浓度或参与硫酸软骨素链合成的三种糖基转移酶（半乳糖基转移酶-I、N-乙酰半乳糖胺基转移酶和葡萄糖醛酸基转移酶-II）的量没有可检测到的变化。在放线菌酮存在下形成的糖胺聚糖链的大小比对照培养物中形成的链大，而在苄基β-D-木糖苷存在下合成的链始终较小，与放线菌酮的存在无关。这些结果表明，在研究硫酸软骨素生物合成作为完全独立于蛋白聚糖核心蛋白合成的事件时，必须谨慎使用β-D-木糖苷，并且它们还表明核心蛋白可能参与硫酸软骨素合成酶的激活。
