水稻Rubisco亚基结合蛋白的纯化及性质研究

Puritication and Propeties of Ribulose Bisohosphate Carbxylase Subunit Binding Protein in Oryza sativa

以水稻叶片为材料，经热处理、硫酸铵分部，采用了ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅｆａｓｔｆｌｏｗ和Ｓｅｐｈａｃｒｙｌ－３００层析，阶段和线性洗脱相结合的步骤，纯化了Ｒｕｂｉｓｃｏ亚基结合蛋白，其得率比前人报道的方法提高了１倍．纯化的Ｒｕｂｉｓｃｏ亚基结合蛋白的式量为７０８０００，亚基式量为５８９００，故为十二聚体．该蛋白在紫外区有２个吸收峰，分别为２１２．６ｎｍ和２７９．４ｎｍ．该蛋白的氨基酸组分分析表明，含较多的苯丙氨酸，酸性氨基酸和碱性氨基酸的比例为１∶１．３３．

Rice leaves were ground and extracted with 100 mmol/l Tris-HCl(pH8.0) at 0℃ containing 2mM EDTA·2Na and 5% Gly.The crude extract was heated at 55℃ for 10min,then centrifuged at 12 000×g for 20min at 4℃.The superanatant was fractionated by 35% to 60% saturation(NH 4) 2SO 4.The precipitate was dissolved in a Small wolumn, desalted with Sephadex G-50 column and then loaded onto a DEAE-Sepharose Fast Flow Column,The column was washed consecutively with 0.1mol/L NaCl to remove some Rubisco,then washed with a linear gradient of 0.1～0.4 mol/L NaCl.The BP-enriched fractions were pooled and a 70% saturation (NH 4) 2SO 4 precipitate was prepared. The precipitate was redissolved in a small volumn and loaded onto a Sephacryl-300 colum.The BP peak fractions were collected and reprecipitated with (NH 4) 2SO 4(Tablel). The protein exhibited two absorption peaks in ultraviolet both at 212.6nm and at 279.4nm. The protein has a molecular mass about 708 000 and is composed of twelve equal subunits. The molecular mass of the subunit has been found to be 58 900. The protein contains more phenylalanine than those of amid acids and is stable between pH7.64 and 8.0. The protein was stabel by treated with high ion concertation of KCl too.