Granda S, Hustedt H, Flossdorf J, Kula M R
Mol Cell Biochem. 1979 Apr 2;24(3):175-81. doi: 10.1007/BF00220736.
A procedure for the large-scale isolation of leucyl-tRNA synthetase from E. cole MRE 600 is described: The enzyme was purified about 320-fold to homogeneity by precipitation with cetyl-trimethyl-ammonium bromide, two consecutive chromatographies on DEAE-cellulose and three on hydroxyapatite with an over-all yield of 4%. The molecular weight of leucyl-tRNA synthetase from E. coli MRE 600 was found to be 99 000 daltons. Bindings studies by ultracentrifugation and equilibrium partition showed that the enzyme binds leucine, leucyl-adenylate and tRNA Leu, each in a 1 : 1 stoichiometry. For ATP only a very weak binding to the enzyme could be observed, which did not allow the evaluation of the complex stoichiometry. The presence of ATP was not required for the binding of leucine or tRNA to leucyl-tRNA synthetase from E. coli MRE 600.
本文描述了从大肠杆菌MRE 600中大规模分离亮氨酰 - tRNA合成酶的方法：通过用十六烷基三甲基溴化铵沉淀、在DEAE - 纤维素上连续进行两次色谱分离以及在羟基磷灰石上进行三次色谱分离，将该酶纯化至约320倍的同质性，总产率为4%。发现来自大肠杆菌MRE 600的亮氨酰 - tRNA合成酶的分子量为99000道尔顿。通过超速离心和平衡分配进行的结合研究表明，该酶以1:1的化学计量比结合亮氨酸、亮氨酰腺苷酸和tRNA Leu。对于ATP，仅观察到与该酶的非常弱的结合，这使得无法评估复合物的化学计量比。ATP的存在对于亮氨酸或tRNA与来自大肠杆菌MRE 600的亮氨酰 - tRNA合成酶的结合不是必需的。
