Studies on the origin of eukaryotes-Functional analyzes of ribosomes of microsporidia

为了研究真核生物的进化起源，我们尝试建立了一个由包括家蚕微孢子虫在内的一些微孢子虫核糖体组成的体外蛋白质合成系统，该系统被认为在真核生物的早期阶段分化。家蚕新孢子具有与原核生物相似的核糖体沉降特性，从休眠孢子和人工孵化的孢子中提取细胞粗提物。通过差速离心从提取物中回收核糖体，并在体外蛋白质合成系统中与可溶性酶级分孵育。该系统含有外源性添加的聚（U）、^3 H-Phe和ATP再生系统。体外蛋白质合成量的测定是通过计算孵育后热三氯乙酸不溶部分的放射性来进行的，但由于家蚕核糖体和/或酶部分活性差，所有建立该系统的尝试都失败了。添加大肠杆菌、大鼠肝和家蚕中肠酶组分对酶活性无影响。氨酰-tRNA的合成，然而，发生正常的系统从N. bombycis。这些结果可能表明，在家蚕核糖体的制备过程中存在一些抑制因子。

In order to investigate the evolutionary origin of eukaryotes, we attempted to develop an in vitro protein synthesizing system consisting of ribosomes from some microsporidia including Nosema bombycis which is believed to diverge at early stage of eukaryotes. N.bombycis is known to have ribosomes whose sedimentation property is very similar to that of prokaryotes.Crude cellular extract was prepared from both dormant and artificially hatched spores. Ribosomes was recovered from the extract by differential centrifugation and incubated with the soluble enzyme fraction in an in vitro protein synthesizing system. The system contained exogenously added poly (U), ^3H-Phe and an ATP-regenarating system. The amount of in vitro protein synthesis was measured by counting the radioactivity of hot trichloroacetic acid-insoluble fraction after incubation.All the attempt to establish the system was failed because of poor activity of ribosomes and/or enzyme fraction of N.bombycis. Supplementation of the enzyme fraction of Ecsherichia coli, rat liver and midgut of silkworm had no effect on the activity. Aminoacyl-tRNA synthesis, however, took place normally in the system from N.bombycis. These results may indicate that some inhibitory factors are present in the preparation of N.bombycis ribosomes.