Triton X-114 phase fractionation of membrane proteins of the cyanobacterium Anacystis nidulans R2.
Triton X-114 phase fractionation of membrane proteins of the cyanobacterium Anacystis nidulans R2.
复制标题
Triton X-114 相分离蓝藻 Anacystis nidulans R2 的膜蛋白。
DOI：
10.1016/0003-9861(84)90269-8
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发表时间：
1984
影响因子：
3.9
通讯作者：
Sherman,LA
中科院分区：
文献类型：
--
作者：
Bricker,TM;Sherman,LA
利用Triton X-114相分离技术对蓝细菌Anacystis nidulansR2的类囊体多肽进行了分析。Bordier（1981）J.Biol.Chem.256，1604 - 1607，如T. M. Bricker和L. a.谢尔曼（1982）FEBS Lett.149，197 - 202]。在该方法中，具有广泛疏水区域的多肽（即，内在蛋白质）与Triton X-114形成混合胶束，并通过混合Triton X-114-内在蛋白质胶束的温度介导沉淀与外在蛋白质分离。相分离后的多肽模式是高度互补的，与62所观察到的110多肽组分分配到Triton X-114富集馏分。鉴定的多肽分馏到Triton X-114相包括脱辅基蛋白的光系统I和II，细胞色素f和b6，和除草剂结合蛋白。鉴定的多肽分馏到Triton X-114耗尽（水）相包括大和小的RuBp羧化酶，细胞色素c550和c554，和铁氧还蛋白亚基。酶法放射性碘标记的光合膜，然后Triton X-114相分馏允许直接识别的内在多肽成分，具有表面暴露的区域容易受到放射性碘标记。这些多肽中最突出的是与光系统II相关的34 kDa组分。这一阶段的分配程序已特别有助于澄清的身份的膜相关的细胞色素，和光系统II组件。当与表面探测技术相结合时，该程序在鉴定具有表面暴露结构域的内在蛋白质方面非常有用。相分离，结合特定的膜成分和复合物的分离，允许许多重要的内源性类囊体膜蛋白的A。nidulansR2.
The thylakoid polypeptides of the cyanobacteriumAnacystis nidulansR2 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 andb6, 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, cytochromesc550andc554, 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 ofA. nidulansR2.