AFFINITY LABELLING OF E.coil LEUCYL-tRNA SYNTHETASE WITH 3'-OXIDIZED tRNA~(Leu)

• 作 者: 黄守廷 林胜祥 施建平 王应睐
• 机 构: Shanghai Institute of Biochemistry,Academia Sinicarrrrrrrrn,Shanghai Institute of Biochemis
• 出 处: Science in China,Ser.B
• 发表日期: 1987年12期
• 关键词: tRNA labelling digestion subsequent specifically restored inhibited oxidized syn

The E.coli leucyl-tRNA synthetase (E. C. 6. 1. 1. 4) was specifically labelled with 3’-oxidized tRNA~(Leu) (tRNA_(ox)~(Leu)).The procedure involves a Schiff’s base formation and its subsequent reduction by sodium cyanoborohydride. Stoichiometric inactivation of aminoacylation was achieved with the incorporation of 1 mol of tRNA_(ox)~_(Leu) per mol LeuRS.On the other hand, the amino acid activation activity of LeuRS-tRNA_(ox) complex was partially inhibited. After extensive digestion of the complex by pancreatic ribonuclease, the amino acid activation activity was fully recovered, while the aminoacylation activity was not restored at all.The results suggest that the active sites responsible for the activities of LeuRS correlate with each other as well as differ from each other.

The E. coli leucyl-tRNA synthetase (EC 6.1.4) was specifically labeled with 3’-oxidized tRNA ~ (Leu) (tRNA - (Leu)). The procedure involves a Schiff’s base formation and the subsequent reduction by sodium cyanoborohydride. Stoichiometric inactivation of aminoacylation was achieved with the incorporation of 1 mol of tRNA_ (ox) ~_ (Leu) per mol LeuRS.On the other hand, the amino acid activation activity of LeuRS-tRNA_ (ox) complex was partially inhibited. After extensive digestion of the complex by pancreatic ribonuclease, the amino acid activation activity was fully recovered, while the aminoacylation activity was not restored at all. The results suggest that the active sites responsible for the activities of LeuRS correlate with each other as well as differ from each other.