title: In vitro reassembly of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase from fully denatured subunits.
abstract: 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 Mg2+ were unnecessary for in vitro reassembly of Rubisco, and Mg2+ inhibited the reassembly process. The reassembly was weakened when ATP, Mg2+ and K+ existed together in the reassembly process.
authors.affiliation: 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.
authors.forename: Zhen-Hua
authors.initials: ZH
authors.lastname: Yong
authors.forename: Gen-Yun
authors.initials: GY
authors.lastname: Chen
authors.forename: Jiao-Nai
authors.initials: JN
authors.lastname: Shi
authors.forename: Da-Quan
authors.initials: DQ
authors.lastname: Xu
mesh_terms: D018834:Chaperonin 60 / Q000502:physiology; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology; D011489:Protein Denaturation; D020673:Protein Renaturation; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000502:physiology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology*
chemical_list: D018834:Chaperonin 60; D018840:HSP70 Heat-Shock Proteins; D021122:Protein Subunits; D012273:Ribulose-Bisphosphate Carboxylase