In Vitor Ressembly Of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase And The Function Of Chaperone

As the media of CO₂ entrance to organism and inorganic carbon transformation to organic carbon, ribulose-1,5-bisphosphat carboxylase/oxygenase (Rubisco) is crucial for all lives on earth. And it is obviously important for photosynthetic research. However, Rubisco from higher plant is inefficient. Although many efforts have been made in molecular genetic engineering on Rubisco in higher plant to increase catalytic efficiency, no success has been reported. One of the biggest obstacles is lacking an understanding of Rubisco assembly mechanism. Obviously, further knowledge of Rubisco assembly mechanism might assist the improvement of Rubisco. This research work is mainly focused on Rubisco assembly, especially the function of chaperone on Rubisco assembly. The main results could be concluded in following two parts.1 In vitro reassembly of tobacco Rubisco from fully denatured subunitsIt has been generally proved impossible to reassemble higher plant Rubisco from fully denatured subunits in vitro, because fully denatured Rubisco large subunit (LS) is apt to precipitate when denaturant is removed by common method– direct dilution or one-step dialysis. In this study reported here, the problem of precipitation was resolved by an improved method– gradual dialysis, using which the concentration of denaturant decreased gradually. However, fully denatured Rubisco subunits still could not be reassembled into holoenzyme after gradual dialysis unless chaperonin 60 (cpn60) 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 (hsp70) existed in reassembly assay. 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 reassembly assay.2 The species-specificity of cpn 60 for Rubisco reassembly in vitroThe in vitro reassembly of Rubisco with the help of cpn60 from different species was detected by Native-PAGE. Tobacco (and rice) cpn60 could assist the in vitro reassembly of Rubisco from Porphyridium sp.. However, cpn60 from spinach and pea could not assist the reassembly, and they could not assist the reassembly of Rubisco from tobacco, spinach and pea, indicating that there is a species-specificity of cpn60 for Rubisco reassembly. The analysis of amino acid residue sequences of cpn60α-subunits showed that the 4, 10, 91, 173, 181, 306, 298, 471, 533 and 540th amino acid residues were different between tobacco (rice) and pea. Also, the analysis of amino acid residue sequences of cpn60β-subunits showed that the 171, 180, 238, 241, 270, 299, 300, 353, 370, 396, 402, 407, 428, 479, 489, 493, 534, 542, 567, 575, 578 and 588th amino acid residues were different between rice and pea. Moreover, plants used in this analysis could be divided into two groups based on the differences in amino acid kind in each sites mentioned above. These differences may be the structural basis of cpn60 species-specificity.