Souciet G, Dietrich A, Colas B, Razafimahatratra P, Weil J H
J Biol Chem. 1982 Aug 25;257(16):9598-604.
The chloroplast leucyl-tRNA synthetase from Phaseolus vulgaris was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, hydroxylapatite, and phosphocellulose. Finally, the pure enzyme was obtained after affinity chromatography on Blue Sepharose CL-6B using specific elution with a pure Escherichia coli tRNALeu isoacceptor. The specific activity of chloroplast leucyl-tRNA synthetase (1550 units/mg) is the highest ever obtained for a higher plant aminoacyl-tRNA synthetase. The purified enzyme has an optimal pH of 9.0 and Km values are, respectively, 2.6 X 10(-6) M for unfractionated E. coli tRNA, 0.85 X 10(-6) M for E. coli tRNA5Leu, 1.8 X 10(-4) M for ATP, and 1.4 X 10(-5) M for L-leucine. Chloroplast leucyl-tRNA synthetase is a large monomer which has a Mr of 122,000 as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate and urea and by gel filtration. Determination of Stokes radius, diffusion coefficient, and frictional ratio suggests that the enzyme structure is rather compact. The amino acid composition shows a relatively large proportion of apolar residues. Specific antibodies were raised in rabbits against the pure chloroplast leucyl-tRNA synthetase.
菜豆叶绿体亮氨酰 - tRNA合成酶通过硫酸铵沉淀以及在DEAE - 纤维素、羟基磷灰石和磷酸纤维素上的色谱法进行纯化。最后，使用纯的大肠杆菌亮氨酰tRNA同工受体进行特异性洗脱，在蓝色琼脂糖凝胶CL - 6B上进行亲和色谱后获得了纯酶。叶绿体亮氨酰 - tRNA合成酶的比活性（1550单位/毫克）是高等植物氨酰 - tRNA合成酶中迄今所获得的最高值。纯化后的酶的最适pH为9.0，其米氏常数分别为：对于未分级的大肠杆菌tRNA为2.6×10⁻⁶ M，对于大肠杆菌tRNA⁵Leu为0.85×10⁻⁶ M，对于ATP为1.8×10⁻⁴ M，对于L - 亮氨酸为1.4×10⁻⁵ M。叶绿体亮氨酰 - tRNA合成酶是一种大的单体，在十二烷基硫酸钠和尿素存在下通过聚丙烯酰胺凝胶电泳以及凝胶过滤测定其相对分子质量为122,000。斯托克斯半径、扩散系数和摩擦比的测定表明该酶结构相当紧密。氨基酸组成显示非极性残基的比例相对较大。用纯的叶绿体亮氨酰 - tRNA合成酶对兔子进行免疫以产生特异性抗体。
