Bricker T M, Sherman L A
Arch Biochem Biophys. 1984 Nov 15;235(1):204-11. doi: 10.1016/0003-9861(84)90269-8.
The thylakoid polypeptides of the cyanobacterium Anacystis nidulans R2 were analyzed by Triton X-114 phase fractionation [C. Bordier (1981) J. Biol. Chem. 256, 1604-1607, as adapted for photosynthetic membranes by T.M. Bricker and L.A. Sherman (1982) FEBS Lett. 149, 197-202]. In this procedure, polypeptides with extensive hydrophobic regions (i.e., intrinsic proteins) form mixed micelles with Triton X-114, and are separated from extrinsic proteins by temperature-mediated precipitation of the mixed Triton X-114-intrinsic protein micelles. The polypeptide pattern after phase fractionation was highly complementary, with 62 of the observed 110 polypeptide components partitioning into the Triton X-114-enriched fraction. Identified polypeptides fractionating into the Triton X-114 phase included the apoproteins for Photosystems I and II, cytochromes f and b6, and the herbicide-binding protein. Identified polypeptides fractioning into the Triton X-114-depleted (aqueous) phase included the large and small subunits of RuBp carboxylase, cytochromes c550 and c554, and ferredoxin. Enzymatic radioiodination of the photosynthetic membranes followed by Triton X-114 phase fractionation allowed direct identification of intrinsic polypeptide components which possess surface-exposed regions susceptible to radioiodination. The most prominent of these polypeptides was a 34-kDa component which was associated with photosystem II. This phase partitioning procedure has been particularly helpful in the clarification of the identity of the membrane-associated cytochromes, and of photosystem II components. When coupled with surface-probing techniques, this procedure is very useful in identifying intrinsic proteins which possess surface-exposed domains. Phase fractionation, in conjunction with the isolation of specific membrane components and complexes, has allowed the identification of many of the important intrinsic thylakoid membrane proteins of A. nidulans R2.
通过Triton X-114相分离法[C. Bordier（1981年）《生物化学杂志》256卷，1604 - 1607页，经T.M. Bricker和L.A. Sherman（1982年）改编用于光合膜，《欧洲生物化学学会联合会快报》149卷，197 - 202页]分析了集胞藻6803 R2的类囊体多肽。在此过程中，具有广泛疏水区域的多肽（即内在蛋白）与Triton X-114形成混合胶束，并通过温度介导的混合Triton X-114 - 内在蛋白胶束沉淀与外在蛋白分离。相分离后的多肽图谱具有高度互补性，在观察到的110种多肽组分中，有62种分配到富含Triton X-114的组分中。分配到Triton X-114相的已鉴定多肽包括光系统I和II的脱辅基蛋白、细胞色素f和b6以及除草剂结合蛋白。分配到Triton X-114耗尽（水相）相的已鉴定多肽包括核酮糖-1,5-二磷酸羧化酶的大亚基和小亚基、细胞色素c550和c554以及铁氧还蛋白。光合膜经酶促放射性碘化后再进行Triton X-114相分离，可直接鉴定出具有易被放射性碘化的表面暴露区域的内在多肽组分。其中最突出的多肽是一种与光系统II相关的34 kDa组分。这种相分配方法在阐明膜相关细胞色素和光系统II组分的身份方面特别有帮助。当与表面探测技术结合使用时，该方法对于鉴定具有表面暴露结构域的内在蛋白非常有用。相分离与特定膜组分和复合物的分离相结合，已使得能够鉴定出集胞藻6803 R2许多重要的内在类囊体膜蛋白。
