CHLOROPLAST MOLECULAR CHAPERONE ASSISTED REFOLDING AND RECONSTITUTION OF AN ACTIVE MULTISUBUNIT COUPLING FACTOR CF1 CORE
CHLOROPLAST MOLECULAR CHAPERONE ASSISTED REFOLDING AND RECONSTITUTION OF AN ACTIVE MULTISUBUNIT COUPLING FACTOR CF1 CORE
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DOI：
10.1073/pnas.91.24.11497
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发表时间：
1994-11-22
影响因子：
11.1
通讯作者：
JAGENDORF, AT
中科院分区：
文献类型：
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作者：
CHEN, GG;JAGENDORF, AT
叶绿体偶联因子1(CF1)由5种亚基组成，其化学计量比为α(3)-β(3)-γ-βepsilon本文报道了在一定的生理pH条件下，由尿素变性的亚基重构具有催化活性的α(3)-β(3)-伽马核。已观察到CF1 ATPase活性恢复了约90%。重组是通过使用在大肠杆菌中高表达的亚基，纯化，并在镁ATP，K+，和几个叶绿体分子伴侣的混合物在pH 7.5的存在下结合实现的。伴侣蛋白60和伴侣蛋白24的结合未能重组活性CF1核心，GroEL/GroES对(大肠杆菌伴侣蛋白60/10同源物)也是如此。重组ATPase的性质与天然复合体非常接近，包括纯化的CF1 epsilon亚基对甲醇的可逆抑制，以及对叠氮和天冬氨酸的敏感性。在混合了氨曲毒素抗性和敏感的β亚基的重组过程中，其抑制程度取决于敏感亚基在重组混合物中所占的比例。最后，提出了CF1核α(3)β(3)伽马结构的组装模型。
The chloroplast coupling factor 1 (CF1) is composed of five kinds of subunits with a stoichiometry of alpha(3) beta(3) gamma delta epsilon Reconstitution of a catalytically active alpha(3) beta(3) gamma core from urea-denatured subunits at a physiological pH is reported here. A restoration of approximately 90% of the CF1 ATPase activity has been observed. The reconstitution was achieved by using subunits overexpressed in Escherichia coli, purified, and combined in the presence of MgATP, K+, and a mixture of several chloroplast molecular chaperones at pH 7.5. The combination of chaperonin 60 and chaperonin 24 failed to reconstitute the active CF1 core, as did the GroEL/GroES pair (E. coli chaperonin 60/10 homologues). Characteristics of the reconstituted ATPase were very close to those of the native complex, including methanol-reversible inhibition by the purified epsilon subunit of CF1 and sensitivity to Inhibition by azide and by tentoxin. In reconstitution with a mixture of tentoxin-resistant and -sensitive beta subunits, the extent of inhibition by tentoxin depended on the proportion of sensitive subunits in the reconstitution mixture. Finally, a model for the assembly of the CF1 core alpha(3) beta(3) gamma structure is proposed.