| 摘要: |
| 改进了以往分离纯化Cyt b6 f的方法，进行了海洋绿藻——假根羽藻(Bryopsis corticulans) Cyt b6 f蛋白复合体分离纯的研究。结果表明，该Cytb6f制剂由Cyt f、CytbRieske [Fe-S]蛋白和亚基IV四种主要的多肽亚基以及少量的Ch1. a和类胡萝卜素组成。4个多肽亚基的表观分子量分别为34.8、24.0、18.7及16.7kD，该Cyt b6 f制剂的Cyt b6 f比值接近2.0，其纯度值为9.9 nmolCyt f/mg，其催化电子传递的活性(C10-PQH2?PC)为73e/s。上述分析表明，假根羽藻Cytb6 f在组成、纯度和催化活性方面均与已报道的高等植物、淡水绿藻和光合细菌的Cytb6 f制剂相似。 |
| 关键词: 海洋绿藻，假根羽藻，Cyt b6 f，分离，纯化 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金资助项目, 39890390号和30370347号 |
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| IAOLATION AND PURIFICATION OF CYT b6 f COMPLEX FROM BRYOPSIS CORTICULANS |
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LI Bin-Xing1, MAO Da-Zhang1, GAO Zhen-Pan2, LI Liang-Bi1, KUANG Ting-Yun1
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1.Key Laboratory o f Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Science;2.Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Science
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| Abstract: |
| As a plastoquinol-plastocyanin oxidoreductase,cytochrome b6 f complex plays an important role in electron transfer and energy transduction in photosynthesis. It mediates the linear electron flow between PSII and PSI, catalyzes the cyclic electron flow around PSI, and sets up a transmembrane proton electrochemical potential to support energy to form ATP. In addition, cytochrome b6 f complex is also involved in the regulation of balanced light excitation energy distribution between photosystems since its redox states governs the activation of LHCII kinase. It consists of four major subunits (Cyt f, Cyt b, Rieske iron-sulfur protein, and subunit IV) and four small subunits (pet G, L, M, and N). Besides, about one chlorophyll a molecule and one or less than one carotenoid molecule have been found in each cytochrome b6f monomer.The cytochrome b6f complex has been purified from higher plants, freshwater green algae and cyanobacteria. However, no case has been reported of this complex from marine green alga. In this work, the cytochrome b6f complex was purified from a marine green alga—Bryopsis corticulans successfully by an improved method, its composition and activity were studied in this paper. According to the purification of spinach b6f complex, Cyt b6f was not isolated from Bryopsis corticulans thylakoid. It was shown that most of the b6f complex has gone before last ammonium sulfate fractionati0ns.To solve this problem, ammonium sulfate concentration was adjusted. Although this modification was proved to be useful, some 40—60kD extrinsic proteins still existed. To effectively remove these extrinsic proteins, 2mol/L NaBr membrane washing was repeated after that the extrinsic proteins were removed successfully shown by SDS-PAGE. This modified purification procedures for Bryopsis corticulans b6fcomplex are: Chloroplasts were isolated from 2kg pre-chilled fresh Bryopsis corticulans as regular way. After osmotically broken, the chloroplasts were resuspended in 2mol/L NaBr, l0mmoL/L Tricine-NaOH (pH 8.0), 0.4mol sucrose and the concentration of chloroplasts was diluted to 1mg/ml, then centrifuged at 8000g for 20min. This step (2mol/L NaBr membrane washing) was repeated. Cytochrome b6f extraction was carried out as regular way. The crude b6f complex then precipitated by raising ammonium sulfate concentration from 45%一60% and was resuspended in 30mmol/L Tis-suc(pH 7.0), containing 1% sodium cholate. After dialyzed for 12h against 30mmol/L Tis-suc(pH 7.0), 25 mmol/L sucrose, 10mmol/L NaC1 and 40 μmol/L PMSF,the protein solu—tion was centrifuged at 40000g for 5min. The cytochrome b6f pellet was resuspended to about 20 μmol/L Cytf in 20mmol Tricine-NaOH(pH 8.0), containing 30mmol/L β-OG and 0.5% sodium eholate and was subjected to further ammonium sulfate fractionations. Precipitate formed in fractions from 38% 一45% was the end product of purified cytochrome b6f. All the tests were performed at 4°C under dim light. Using this improved method, a pure and active Cyt b6f was purified from marine green alga Bryopsis corticulas. It was shown that the purity of this complex neared 100% (9.9nmol cyt f/mg)and its activity was 73e/s(C10PQH2?PC). Molecular masses of the four major polypeptides were 34.8kD, 24kD, 18.7kD, and 16.7kD. The ratio of Cyt b6 to Cyt f was 2.02:1. Besides Ch1. a, there are two kinds of carotenoids in this complex and their intensity peaks were around 443.2 and 471.4nm ,438.4 and 465.6nm, respectively. They were different from the carotenoids reported in b6f complex and their absorption spectra were similar to those of α-carotene. Ongoing investigation is in progress. |
| Key words: Marine green alga, Bryopsis corticulans, Cyt b6 f complex, Isolation, Purification |
