Imbault P, Colas B, Sarantoglou V, Boulanger Y, Weil J H
Biochemistry. 1981 Sep 29;20(20):5855-9. doi: 10.1021/bi00523a031.
Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.
通过一系列步骤，包括硫酸铵沉淀以及在羟基磷灰石、二乙氨基乙基纤维素、琼脂糖6B、磷酸纤维素和蓝色葡聚糖-琼脂糖上进行层析，将纤细裸藻叶绿体亮氨酰-tRNA合成酶纯化至同质。纯化后的酶表现出的比活性为1233单位/毫克蛋白质，这是从植物细胞制备的氨酰-tRNA合成酶所获得的最高比活性之一。该酶对L-亮氨酸的表观Km值为8×10⁻⁶M，对ATP为1.3×10⁻⁴M，对tRNALeu为1.3×10⁻⁶M。叶绿体亮氨酰-tRNA合成酶似乎是一种分子量为100000的单体酶。已测定了叶绿体亮氨酰-tRNA合成酶的氨基酸组成。这是首次报道的叶绿体氨酰-tRNA合成酶的氨基酸组成，它显示出相对较大比例的非极性残基，与原核生物氨酰-tRNA合成酶的情况相同。
