Ogata K, Kurahashi A, Ohno R, Takahashi K, Terao K
Institute for Gene Expression, Dobashi Kyoritsu Hospital, Ehime.
J Biochem. 1995 Apr;117(4):750-7. doi: 10.1093/oxfordjournals.jbchem.a124772.
Rat liver cytosol was incubated with a trace amount of rat liver 5SrRNA which was highly labeled at the 3'-end with cytidine 3',5'-[5'-32P]biphosphate, and with [35S]methionine in the presence of ATP mixture, and then with an antibody against ribosomal protein L5. The mixture was analyzed by protein A-Sepharose chromatography. The following results were obtained. (i) The eluate with glycine-HCl buffer (pH 3.0) from the protein A-Sepharose column contained an overlapping peak of 32P- and 35S-radioactivities. In a control experiment using the same amount of 32P-labeled Escherichia coli 5SrRNA with the same specific activity, no fraction of the eluate contained 32P-radioactivity. (ii) The fractions containing both 32P- and 35S-radioactivities from the protein A-Sepharose column were crosslinked by UV irradiation. The products was subjected to PAGE, and RNA in each gel slice was eluted and purified. The fraction containing both 32P- and 35S-radioactivities was present in a region of somewhat higher molecular weight than that of 5SRNP, whereas very low 32P- and 35S-radioactivities were present in this region in the control experiment without UV irradiation. This finding suggested that [35S]methionyl-tRNA interacted with 32P-labeled 5SRNP. (iii) The fraction containing overlapping 32P- and 35S-radioactivities described above was subjected to Sephadex G-150 chromatography. The component containing both radioactivities was distributed in the region corresponding to molecular weights of 10,000 to 250,000 with a peak at about 200,000, suggesting the presence of a complex containing Met-RS (Mr 108,000), 5SRNP (Mr 74,000), and methionyl-tRNA (Mr 25,000). Furthermore, this fraction showed definite Met-RS activity.(ABSTRACT TRUNCATED AT 250 WORDS)
将大鼠肝脏胞质溶胶与微量在3'-末端用胞苷3',5'-[5'-32P]二磷酸高度标记的大鼠肝脏5S rRNA以及在ATP混合物存在下的[35S]甲硫氨酸一起温育，然后与抗核糖体蛋白L5的抗体一起温育。通过蛋白A-琼脂糖凝胶层析分析该混合物。得到以下结果。(i) 从蛋白A-琼脂糖凝胶柱中用甘氨酸-HCl缓冲液（pH 3.0）洗脱的组分含有32P-和35S-放射性的重叠峰。在使用相同比活的相同量的32P标记的大肠杆菌5S rRNA的对照实验中，洗脱液的任何组分都不含32P-放射性。(ii) 来自蛋白A-琼脂糖凝胶柱的含有32P-和35S-放射性的组分通过紫外线照射进行交联。将产物进行聚丙烯酰胺凝胶电泳，洗脱并纯化每个凝胶切片中的RNA。含有32P-和35S-放射性的组分存在于分子量略高于5SRNP的区域，而在没有紫外线照射的对照实验中该区域存在非常低的32P-和35S-放射性。这一发现表明[35S]甲硫氨酰-tRNA与32P标记的5SRNP相互作用。(iii) 对上述含有重叠的32P-和35S-放射性的组分进行Sephadex G-150层析。含有两种放射性的组分分布在分子量为10,000至250,000的区域，峰值约为200,000，表明存在含有甲硫氨酰-tRNA合成酶（Mr 108,000）、5SRNP（Mr 74,0,00）和甲硫氨酰-tRNA（Mr 25,000）的复合物。此外，该组分显示出明确的甲硫氨酰-tRNA合成酶活性。（摘要截断于250字）
