Ogata K, Kurahashi A, Nishiyama C, Terao K
Institute for Gene Expression, Dobashi Kyoritsu Hospital, Ehime, Japan.
Biochim Biophys Acta. 1994 Aug 2;1218(3):388-400. doi: 10.1016/0167-4781(94)90192-9.
A complex containing Thr-RS and His-RS was purified about 1000 to 2000-fold from rat liver cytosol by successive column chromatographies on Sephadex G-200, Phenyl-Sepharose CL-4B, and tRNA-Sepharose. The ratio of the specific activity of Thr-RS and His-RS was relatively constant throughout the purification steps, suggesting that the two synthetases were co-purified as a complex. Chromatographic analyses of the tRNA-Sepharose fraction by Sephadex G-150 column chromatography showed the presence of a hybrid form of the Thr-RS monomer and the His-RS monomer in addition to dimer forms of both enzymes from the pattern of activity of both enzymes. The monomer form of Thr-RS showed high activity comparable to the dimer form and the monomer form of His-RS showed definite activity. An association form of Thr-RS and His-RS dimers was detected by Sephadex G-200 chromatography of rat liver cytosol. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 55SrRNA blot analysis of the tRNA-Sepharose fraction using an antibody against ribosomal protein L5, showed the presence of ribosomal protein L5 in this fraction. These findings suggest that the presence of a 5SRNA-L5 protein complex (5SRNP) in the Thr-RS and His-RS complex. 5SRNP enhanced the activity of Thr-RS in a freshly prepared tRNA-Sepharose fraction. It also enhanced the activity of the rat liver cytosol for the attachment of [3H]threonine to endogenous tRNA. This activity was inhibited by an antibody against protein L5, and the inhibition was reversed by addition of 5SRNP. These results indicate that 5SRNP plays a role as a positive effector of Thr-RS in the complex.
通过在葡聚糖凝胶G - 200、苯基琼脂糖CL - 4B和tRNA - 琼脂糖上连续柱层析，从大鼠肝脏胞质溶胶中纯化出一种含有苏氨酸 - tRNA合成酶（Thr - RS）和组氨酸 - tRNA合成酶（His - RS）的复合物，纯化倍数约为1000至2000倍。在整个纯化步骤中，Thr - RS和His - RS的比活性相对恒定，这表明这两种合成酶作为复合物被共纯化。通过葡聚糖凝胶G - 150柱层析对tRNA - 琼脂糖部分进行色谱分析，从两种酶的活性模式来看，除了两种酶的二聚体形式外，还存在Thr - RS单体和His - RS单体的杂交形式。Thr - RS的单体形式表现出与二聚体形式相当的高活性，His - RS的单体形式表现出一定的活性。通过对大鼠肝脏胞质溶胶进行葡聚糖凝胶G - 200色谱分析，检测到Thr - RS和His - RS二聚体的缔合形式。对从tRNA - 琼脂糖部分制备的RNA进行Northern印迹分析，显示存在55SrRNA；使用抗核糖体蛋白L5的抗体对tRNA - 琼脂糖部分进行印迹分析，显示该部分存在核糖体蛋白L5。这些发现表明在Thr - RS和His - RS复合物中存在5S RNA - L5蛋白复合物（5SRNP）。5SRNP增强了新鲜制备的tRNA - 琼脂糖部分中Thr - RS的活性。它还增强了大鼠肝脏胞质溶胶将[³H]苏氨酸连接到内源性tRNA上的活性。这种活性被抗蛋白L5的抗体抑制，并且通过添加5SRNP可逆转这种抑制作用。这些结果表明5SRNP在复合物中作为Thr - RS的正效应物发挥作用。
