Mizutani T, Narihara T, Hashimoto A
Eur J Biochem. 1984 Aug 15;143(1):9-13. doi: 10.1111/j.1432-1033.1984.tb08331.x.
Seryl-tRNA synthetase was purified 1800-fold from bovine liver extract by ultracentrifugation at 150 000 X g, chromatography on DEAE-cellulose, fractional precipitation with ammonium sulfate, gel chromatography on Sephacryl S-300, adsorption chromatography on hydroxyapatite, affinity chromatography on blue-Sepharose and finally on Matrex gel red A. The relative molecular mass, Mr, in the denatured state was estimated as 87 000 by sodium dodecyl sulfate disc gel electrophoresis; in the active state the Mr, was estimated as 170 000 for the dimeric native enzyme (alpha 2 type) by chromatography on Sephacryl S-300. The amino acid composition of the enzyme was determined. The Km values for ATP and serine were 0.49 mM and 30 microM, respectively. The Km values for tRNASerIGA and tRNASerCmCA were 1.40 microM and 1.25 microM, respectively. Sequences common to the two isoaccepting tRNASer molecules are discussed in relation to the recognition mechanism of the purified seryl-tRNA synthetase.
通过在150 000×g下超速离心、DEAE - 纤维素柱层析、硫酸铵分级沉淀、Sephacryl S - 300凝胶层析、羟基磷灰石吸附层析、蓝色琼脂糖亲和层析以及最后在Matrex gel red A上进行层析，从牛肝提取物中纯化出了1800倍的丝氨酰 - tRNA合成酶。通过十二烷基硫酸钠圆盘凝胶电泳估计其在变性状态下的相对分子质量Mr为87 000；通过Sephacryl S - 300层析估计处于活性状态的二聚体天然酶（α2型）的Mr为170 000。测定了该酶的氨基酸组成。ATP和丝氨酸的Km值分别为0.49 mM和30 μM。tRNASerIGA和tRNASerCmCA的Km值分别为1.40 μM和1.25 μM。结合纯化的丝氨酰 - tRNA合成酶的识别机制，讨论了两种同工受体tRNASer分子共有的序列。
