Roberts W K, Olsen M L
Biochim Biophys Acta. 1976 Dec 13;454(3):480-92. doi: 10.1016/0005-2787(76)90274-4.
Nine aminoacyl-tRNA synthetases from Ehrlich ascites cells were examined with respect to their ability to be isolated as high molecular weight complexes, soluble enzymes, and ribosome-bound enzymes. Several different methods were employed for cell homogenization and enzyme isolation, with particular attention paid to the effects of hypotonic, isotonic, and hypertonic buffers on enzyme isolation. The binding of all synthetases to ribosomes was eliminated if the low ionic strength of the isolation buffer was raised to isotonic levels. In contrast, neither the ionic strength or composition of the buffers, nor the procedures used for cell homogenization or enzyme isolation had any significant effect on the isolation of the high molecular weight synthetase complex. Certain enzymes (lysyl-, methionyl- and isoleucyl-tRNA synthetases) formed very stable complexes and high molecular weight species were the predominant forms of these enzymes under all conditions of cell homogenization and enzyme isolation. Other enzymes (glycyl-, tyrosinyl- and threonyl-tRNA synthetases) formed complexes very weakly, if at all, and always appeared predominately in the soluble enzyme fraction. Isolated soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases did not associate to form significant amounts of complex upon re-isolATION, SUGGESTING THAT A COMPONENT NECESSARY FOR COMPLEX FORMATION WAS MISSING FROM THE SOLUBLE ENZYME FRACTION. However, the soluble forms of these enzymes, but not the glycyl-, tyrosinyl- and threonyl-tRNA synthetases, did for complexes when mixed with ribosomal RNA or polyuridylic acid. Preliminary experiments showed no significant differences between the complexed and soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases with respect to Km values or ability to charge different isoaccepting tRNAs.
对艾氏腹水癌细胞中的九种氨酰 - tRNA合成酶进行了研究，考察它们作为高分子量复合物、可溶性酶和核糖体结合酶被分离出来的能力。采用了几种不同的方法进行细胞匀浆和酶的分离，特别关注低渗、等渗和高渗缓冲液对酶分离的影响。如果将分离缓冲液的低离子强度提高到等渗水平，所有合成酶与核糖体的结合都会被消除。相比之下，缓冲液的离子强度或组成，以及用于细胞匀浆或酶分离的程序，对高分子量合成酶复合物的分离均无显著影响。某些酶（赖氨酰 -、甲硫氨酰 - 和异亮氨酰 - tRNA合成酶）形成非常稳定的复合物，在细胞匀浆和酶分离的所有条件下，高分子量形式都是这些酶的主要形式。其他酶（甘氨酰 -、酪氨酰 - 和苏氨酰 - tRNA合成酶）即使形成复合物也非常弱，并且总是主要出现在可溶性酶部分。重新分离时，分离出的可溶性形式的赖氨酰 -、甲硫氨酰 - 和异亮氨酰 - tRNA合成酶不会缔合形成大量复合物，这表明可溶性酶部分缺少复合物形成所需的一种成分。然而，当这些酶的可溶性形式与核糖体RNA或聚尿苷酸混合时，会形成复合物，但甘氨酰 -、酪氨酰 - 和苏氨酰 - tRNA合成酶不会。初步实验表明，赖氨酰 -、甲硫氨酰 - 和异亮氨酰 - tRNA合成酶的复合形式和可溶性形式在Km值或对不同同工受体tRNA进行氨酰化的能力方面没有显著差异。
