来自面包酵母的精氨酰 - tRNA合成酶。纯化及某些性质

Arginyl-tRNA synthetase from baker's yeast. Purification and some properties.

作者信息

Gangloff J, Schutz A, Dirheimer G

出版信息

Eur J Biochem. 1976 May 17;65(1):177-82. doi: 10.1111/j.1432-1033.1976.tb10403.x.

DOI:10.1111/j.1432-1033.1976.tb10403.x

Abstract

Arginyl-tRNA synthetase from baker's yeast (Saccharomyces cerevisiae, strain 836) was obtained pure by a large-scale preparative method, which involves four chromatographic columns and one preparative polyacrylamide gel electrophoretic step. The enzyme has a high specific activity (9000 U/mg) and consists of a single polypeptide chain of molecular weight approximately 73000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Amino acid analysis of the enzyme permitted calculation of the absorption coefficient of arginyl-tRNA synthetase (A(1 mg/ml 280 nm)=1.26). Concerning kinetic parameters of the enzyme we found the following Km values: 0.28 muM, 300 muM, 1.5 muM for tRNA(Arg III), ATP and arginine in the aminoacylation reaction, and 1400 muM, 2.5 muM, and 50 muM for ATP, arginine and PP(i) in the ATP-PP(i) exchange reaction. Arginyl-tRNA synthetase required tRNA(Arg III) to catalyse the ATP-PP(i) exchange reaction.

摘要

通过一种大规模制备方法获得了来自面包酵母（酿酒酵母，836菌株）的精氨酰 - tRNA合成酶的纯品，该方法包括四个色谱柱和一个制备型聚丙烯酰胺凝胶电泳步骤。该酶具有高比活性（9000 U/mg），由一条分子量约为73000的单多肽链组成，这是在十二烷基硫酸钠存在下通过聚丙烯酰胺凝胶电泳测定的。对该酶的氨基酸分析使得能够计算精氨酰 - tRNA合成酶的吸收系数（A(1 mg/ml 280 nm)=1.26）。关于该酶的动力学参数，我们在氨基酰化反应中发现tRNA(Arg III)、ATP和精氨酸的Km值分别为0.28 μM、300 μM、1.5 μM，在ATP-PP(i)交换反应中ATP、精氨酸和PP(i)的Km值分别为1400 μM、2.5 μM和50 μM。精氨酰 - tRNA合成酶需要tRNA(Arg III)来催化ATP-PP(i)交换反应。