In Vitro Reassembly of Tobacco Ribulose-1,5-bisphosphate Carboxylase/Oxygenase from Fully Denatured Subunits  

作　　者：Zhen-Hua YONG Gen-Yun CHEN Jiao-Nai SHI Da-Quan XU 

机构地区：[1]Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Graduate School of the Chinese Academy of Sciences, Shanghai 200032, China

出　　处：《Acta Biochimica et Biophysica Sinica》2006年第10期737-745,共9页生物化学与生物物理学报（英文版）

基　　金：This work was supported by the grants from the National Natural Science Foundation of China (No.30400082);the National Basic Research Program of China (No.2005CB121106)

摘　　要：It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/ oxygenase （Rubisco） from fully denatured subunits in vitro in higher plant, because large subunit of fully denatured Rubisco is liable to precipitate when the denaturant is removed by common methods of direct dilution and one-step dialysis. In our experiment, the problem of precipitation was resolved by an improved gradual dialysis method, which gradually decreased the concentration of denaturant. However, fully denatured Rubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60 was added. The restored activity of reassembled Rubisco was approximately 8% of natural enzyme. The quantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassembly process. ATP and Mg^2＋ were unnecessary for in vitro reassembly of Rubisco, and Mg^2＋ inhibited the reassembly process. The reassembly was weakened when ATP, Mg^2＋ and K^＋ existed together in the reassembly process.It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/ oxygenase （Rubisco） from fully denatured subunits in vitro in higher plant, because large subunit of fully denatured Rubisco is liable to precipitate when the denaturant is removed by common methods of direct dilution and one-step dialysis. In our experiment, the problem of precipitation was resolved by an improved gradual dialysis method, which gradually decreased the concentration of denaturant. However, fully denatured Rubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60 was added. The restored activity of reassembled Rubisco was approximately 8% of natural enzyme. The quantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassembly process. ATP and Mg^2＋ were unnecessary for in vitro reassembly of Rubisco, and Mg^2＋ inhibited the reassembly process. The reassembly was weakened when ATP, Mg^2＋ and K^＋ existed together in the reassembly process.

关 键 词：RUBISCO reassembly gradual dialysis chaperonin 60 Hsp70 

分 类 号：Q949[生物学—植物学]