Siddiqui F A, Yang D C
Biochim Biophys Acta. 1985 Apr 5;828(2):177-87. doi: 10.1016/0167-4838(85)90055-x.
Methionyl-tRNA synthetase occurs free and as high-molecular-weight multi-enzyme complexes in rat liver. The free form is purified to near homogeneity by conventional column chromatography and affinity chromatography on tRNA-Sepharose. The native molecular weight of free methionyl-tRNA synthetase is 64 500, based on its sedimentation coefficient of 4.5 S and Stokes radius of 33 A. The free methionyl-tRNA synthetase apparently belongs to alpha-type subunit structure, since the subunit molecular weight is 68 000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Methionyl-tRNA synthetase is dissociated from the high-molecular-weight synthetase complex by controlled trypsinization, according to Kellermann, O., Viel, C. and Waller, J.P. (Eur. J. Biochem. 88 (1978) 197-204). The dissociated, free methionyl-tRNA synthetase is subsequently purified to near homogeneity. The subunit structure of dissociated methionyl-tRNA synthetase is identical to that of endogenous free methionyl-tRNA synthetase. Anti-serum raised against Mr 104 000 protein in the synthetase complex, specifically inhibited methionyl-tRNA synthetase in both the free and the high-molecular-weight forms to the same extent. These results suggest that the occurrence of multiple forms of methionyl-tRNA synthetases in mammalian cells may, in part, be due to proteolytic cleavage.
甲硫氨酰 - tRNA合成酶在大鼠肝脏中以游离形式和高分子量多酶复合物的形式存在。通过常规柱色谱和tRNA - 琼脂糖亲和色谱将游离形式纯化至接近均一。根据其4.5 S的沉降系数和33 Å的斯托克斯半径，游离甲硫氨酰 - tRNA合成酶的天然分子量为64500。游离甲硫氨酰 - tRNA合成酶显然属于α型亚基结构，因为通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳测定的亚基分子量为68000。根据凯勒曼、O.、维埃尔、C.和沃勒、J.P.（《欧洲生物化学杂志》88（1978）197 - 204）的方法，通过可控的胰蛋白酶消化将甲硫氨酰 - tRNA合成酶从高分子量合成酶复合物中解离出来。随后将解离的游离甲硫氨酰 - tRNA合成酶纯化至接近均一。解离的甲硫氨酰 - tRNA合成酶的亚基结构与内源性游离甲硫氨酰 - tRNA合成酶的亚基结构相同。针对合成酶复合物中分子量为104000的蛋白质产生的抗血清，同样程度地特异性抑制游离形式和高分子量形式的甲硫氨酰 - tRNA合成酶。这些结果表明，哺乳动物细胞中甲硫氨酰 - tRNA合成酶多种形式的出现可能部分归因于蛋白水解切割。
