Honda A, Kazuno S, Mori Y, Kimata K, Suzuki S
Department of Biochemistry, Tokyo College of Pharmacy, Japan.
Biochem J. 1987 Sep 15;246(3):745-53. doi: 10.1042/bj2460745.
Since administration of 6-aminonicotinamide (10 micrograms) to day-4 chick embryos in ovo was shown to induce micromelial limbs, biosynthesis of cartilage-characteristic proteoglycan-H (PG-H) as an important index of limb chondrogenesis was examined in day-7 normal and micromelial hind limbs by biochemical and immunological methods. (1) Metabolic labelling of the micromelial limbs with [6-3H]glucosamine and either [35S]sulphate or [35S]methionine, followed by analyses of labelled PG-H by glycerol density-gradient centrifugation under dissociative conditions, showed a marked reduction in the PG-H synthesis. (2) PG-H synthesized by the micromelial limbs was much lower than that synthesized by the normal limbs in the biosynthetic ratio of chondroitin sulphate to keratan sulphate and glycoprotein-type oligosaccharide, although no significant difference was observed in the immunological properties of these proteoglycans. (3) The degree of sulphation of chondroitin sulphates of PG-H was lowered in the micromelial limbs as judged by the increase of unsulphated disaccharide (delta Di-OS) released by chrondroitinase ABC digestion, although there were no significant differences between the normal and the micromelial limbs in the average molecular size (Mr = 38,000) of labelled chondroitin sulphates of PG-H. (4) Addition of beta-D-xyloside, an artificial initiator for chondroitin sulphate synthesis, to the micromelial limbs in culture recovered the incorporation of labelled glucosamine into chondroitin sulphate to that comparable with the normal control with beta-D-xyloside, although the incorporation of [35S]sulphate was lower in the micromelia than in the control with beta-D-xyloside. These results suggest that the reduction in the biosynthesis of the PG-H as well as the production of altered forms of PG-H induced by 6-aminonicotinamide during a critical period of limb morphogenesis may be an important factor for the micromelia.
由于已证明在鸡胚孵化第4天向其体内注射6-氨基烟酰胺（10微克）可诱导肢体微小畸形，因此通过生化和免疫方法，在第7天正常和微小畸形的后肢中，研究了作为肢体软骨形成重要指标的软骨特征性蛋白聚糖-H（PG-H）的生物合成。（1）用[6-³H]葡萄糖胺和[³⁵S]硫酸盐或[³⁵S]甲硫氨酸对微小畸形肢体进行代谢标记，然后在解离条件下通过甘油密度梯度离心分析标记的PG-H，结果显示PG-H合成显著减少。（2）微小畸形肢体合成的PG-H在硫酸软骨素与硫酸角质素以及糖蛋白型寡糖的生物合成比例上远低于正常肢体，尽管这些蛋白聚糖的免疫特性未观察到显著差异。（3）通过软骨素酶ABC消化释放的未硫酸化二糖（δDi-OS）增加来判断，微小畸形肢体中PG-H的硫酸软骨素硫酸化程度降低，尽管PG-H标记的硫酸软骨素的平均分子大小（Mr = 38,000）在正常和微小畸形肢体之间没有显著差异。（4）在培养的微小畸形肢体中添加硫酸软骨素合成的人工引发剂β-D-木糖苷，可使标记的葡萄糖胺掺入硫酸软骨素的量恢复到与添加β-D-木糖苷的正常对照相当的水平，尽管微小畸形肢体中[³⁵S]硫酸盐的掺入量低于添加β-D-木糖苷的对照。这些结果表明，在肢体形态发生的关键时期，6-氨基烟酰胺诱导的PG-H生物合成减少以及PG-H改变形式的产生可能是导致肢体微小畸形的重要因素。
