Role of the Chlamydomonas reinhardtii Coupling Factor 1 γ-Subunit Cysteine Bridge in the Regulation of ATP Synthase(*)
Role of the Chlamydomonas reinhardtii Coupling Factor 1 γ-Subunit Cysteine Bridge in the Regulation of ATP Synthase(*)
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莱茵衣藻偶联因子 1 γ-亚基半胱氨酸桥在 ATP 合酶调节中的作用(*)
DOI：
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发表时间：
1995
影响因子：
4.8
通讯作者：
B. Selman
中科院分区：
文献类型：
--
作者：
S. Ross;Michael Ruogu Zhang;B. Selman
偶联因子1的γ亚基含有一个半胱氨酸桥，被认为参与了酶活性的氧化还原控制。为了验证该二硫键的调控意义，对莱茵衣藻进行了遗传转化实验。将−-亚基缺失的莱茵梭菌atpC1(Nit1-305，Cw 15，mtγ)转化为γ-亚基，并与位于Cys198和Cys204之间的半胱氨酸桥上的氨基酸取代的DNA亚基构建互补。通过表型选择、Northern印迹分析、逆转录聚合酶链式反应和cDNADNA测序证实了成功的互补。在二硫苏糖醇(DTT)存在和不存在的情况下，测定可溶性酶的CF1ATPase活性。突变的CF1酶对DTT没有影响，但观察到野生型酶的活性增加。在体外，依赖吩嗪甲硫酸盐的光磷酸化实验表明，野生型CF1在25 mM DTT存在下表现出2倍的刺激，而每个突变酶都具有DTT不依赖的活性。生长测量表明，尽管没有调节的二硫化物/二硫醇，突变菌株的生长动力学与野生型相同。本研究为阐明γ亚单位参与体内氧化还原合成的调控提供了证据。这项工作也是莱茵梭菌利用突变基因来补充已知缺陷进行稳定核转化的第一个证明。
The γ-subunit of coupling factor 1 (CF1) contains a cysteine bridge that is thought to be involved in the redox control of enzymatic activity. In order to test the regulatory significance of this disulfide bond, genetic transformation experiments with Chlamydomonas reinhardtii were performed. C. reinhardtii strain atpC1 (nit1-305, cw 15, mt−), which is null for the γ-subunit, was transformed and complemented with γ-subunit constructs containing amino acid substitutions localized to the cysteine bridge between Cys198and Cys204. Successful complementation was confirmed by phenotypic selection, Northern blot analysis, reverse transcription polymerase chain reaction, and cDNA sequencing. CF1ATPase activities of the soluble enzymes were measured in the presence and absence of dithiothreitol (DTT). Mutant CF1enzymes showed no effect of DTT although increased activity was observed for the wild-type enzyme. In vitro, phenazine methosulfate-dependent photophosphorylation assays revealed that wild-type CF1exhibits a 2-fold stimulation in the presence of 25 mM DTT, whereas each of the mutant enzymes has activities that are DTT-independent. Growth measurements indicated that despite the absence of a regulatory disulfide/dithiol, the mutant strains grew with the same kinetics as wild type. This study provides evidence to illustrate the involvement of the γ-subunit in the redox regulation of ATP synthesis in vivo. This work is also the first demonstration in C. reinhardtii of stable nuclear transformation using mutated genes to complement a known defect.
DOI：
10.1016/s0021-9258(17)39870-8
发表时间：
1984-06
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Stuart R. Ketcham;James W. Davenport;Kurt WarnckelI;R. E. Mccarty
通讯作者：
Stuart R. Ketcham;James W. Davenport;Kurt WarnckelI;R. E. Mccarty
莱茵衣藻 Ac-208 的质体蓝素缺陷表型是由编码前质体蓝素的核基因中的移码突变造成的。
DOI：
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发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Quinn,J;Li,HH;Singer,J;Morimoto,B;Mets,L;Kindle,K;Merchant,S
通讯作者：
Merchant,S