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

为了探索真核生物的进化起源，我们试图建立一个由微孢子虫核糖体组成的体外蛋白质合成系统，其中包括被认为在真核生物早期发散的家蚕微孢子虫。已知的家蚕核糖体具有与原核生物非常相似的沉降性。通过差速离心法从提取物中回收核糖体，并与可溶性酶组分在体外蛋白质合成系统中孵育。该体系含有外源添加的Poly(U)、~3H-Phe和一个ATP再生系统。体外蛋白质合成量通过计数孵育后热的三氯乙酸不溶组分的放射性来测量。由于核糖体和/或酶组分的活性较低，建立该系统的所有尝试都失败了。添加大肠杆菌、大鼠肝脏和家蚕中肠的酶组分对酶活性无影响。然而，氨酰-tRNA的合成通常是在家蚕的系统中进行的。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.