摘要
用^(35)S-Met在照光下与豌豆完整叶绿体保温,显示新合成的标记的RubisCO大亚基与结合蛋白形成一复合物,经ATP处理后解离为结合蛋白亚基,同时释放出的标记的RubisCO大亚基参与了RubisCO的装配。豌豆叶片提取液经热处理,硫酸铵分部,DEAE-Sepharose fast flow和Sephacryl S-300柱层析在ND-PAGE,SDS-PAGE上显示为一条带,估计纯度达90％以上,得率比以前报道的高12倍。纯化的结合蛋白表面巯基数经测定为12±1个,总巯基数为36±1个。远紫外CD光谱具有典型的α-螺旋结构的光谱特性,α-螺旋度为39％。此外,以纯化的豌豆结合蛋白制备了多克隆抗体。琼脂糖双扩散实验显示,结合蛋白的抗体与结合蛋白产生一条沉淀线,而与豌豆的RubisCO无沉淀反应,这表明所得到的抗体是高度专一的。
Pea (Pisum sativum) leaves were ground and extracted with 20 mmol/L Tris-HCl (pH 7.8) at 4℃ containing 5 mmol / L mercaptoethanol. The extract was heated at 50℃ for 10 min., then centrifuged at 20000×g for 15 min at 4℃. The supernatant was fractionated by 33% to 70% saturation (NH_4)_2SO_4. The precipicate was dissolved in a small volume, desalted with Sephadex G-50 column, and then loaded onto a DEAD-Sepharose Fast Flow column (2.1×28cm). The column was washed consecutively with 0.1 and 0.2 mol / L NaCl to remove most of the RubisCO, then washed with a linear gradient of 0.2～0.4 mol/L NaCl. The BPenriched fractions were pooled, and a 70% saturation (NH_4)_2SO_4 precipitate was prepared. The precipitate was redissolved in a small volume and loaded onto a Sephacryl S-300 column (1.8×70 cm). The BP peak fractions were collected and reprecipitated with (NH_4)_2SO_4 (Table 1).
BP was examined by both ND PAGE and SDS-PAGE, (Fig. 1, 5), and by BP dissociation caused spccifically by the addition of ATP. It was estimated that the purity of the BP obtained was higher than 90%, and the yields of up to 20 mg/kg fresh weight of leaves were obtained (Table 1), which was 12-fold higher than the previous result.
Radioactive PAGE analysis showed (Fig. 2) that when intact chloroplast was incubated with ^(35)S-Met under illumination, the newly synthesized RubisCO large subunit bound to BP. Addition of ATP to the chloroplast extract could cause the BP to dissociate, and the released large subunits were then assembled into RubisCO.
It was determined that the surface SH groups of BP were 12±1, and the total SH groups were 36±1 (Fig. 6). The intrinsic region CD spectra of BP showed a typical α-helix spectrum (Fig. 7), and it was calculated that there was 39% α-helix in BP. The experiment of heat treatment showed that BP was a heat-stable protein (Table 2).
A highly specific anti-BP serum has been prepared, which gave a single precipitin band with BP, by immunodiffusion tests (Fig. 8), but no precipitin band with RubisCO.
关键词
豌豆
纯化
特性
RUBISCO
RubisCO subunit binding protein
purification
assembly
characterization
