Duhe R J, Selman B R
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Biochim Biophys Acta. 1990 May 15;1017(1):70-8. doi: 10.1016/0005-2728(90)90180-c.
The chloroplast coupling factor 1 complex (CF1) contains an epsilon-subunit which inhibits the CF1 ATPase activity. Chloroform treatment of Chlamydomonas reinhardtii thylakoid membranes solubilizes only forms of the enzyme which apparently lack the delta-subunit. Four interrelated observations are described in this paper. (1) The dithiothreitol- (DTT) induced ATPase activation of CF1(-delta) and the DTT-induced formation of a physically resolvable CF1(-delta,epsilon) from the CF1(-delta) precursor are compared. The similar time-courses of these two phenomena suggest that the dissociation of the epsilon-subunit is an obligatory process in the DTT-induced ATPase activation of soluble CF1. (2) The reversible dissociation of the epsilon-subunit of the CF1 is demonstrated by the exchange of subunits between distinguishable oligomers. 35S-labelled chloroplast coupling factor 1 lacking the delta and epsilon subunits [CF1(-delta,epsilon)] was added to a solution of non-radioactive coupling factor 1 lacking only the delta subunit [CF1(-delta)]. After separation of the two enzyme forms, via high resolution anion-exchange chromatography, radioactivity was detected in the chromatographic fractions containing CF1(-delta). (3) epsilon-deficient CF1 can be resolved from DTT pretreated epsilon-containing CF1 for several days after the removal of DTT. On the other hand, brief incubation of the DTT pretreated epsilon-containing CF1 with low concentrations of o-iodosobenzoate results in chromatographs containing only the peak of epsilon-containing CF1. A simple explanation for this phenomenon is that reduction of CF1 with DTT increases the apparent dissociation constant for the epsilon-subunit to an estimated 3.5 x 10(-8) M (+/- 1.0 x 10(-8) M) from a value of less than or equal to 5 x 10(-11) M for the oxidized enzyme. (4) ATPase activity data show that oxidation of the epsilon-deficient enzyme does not completely inhibit its manifest activity, but oxidation of DTT pre-treated CF1 which contains the epsilon-subunit completely inhibits manifest activity. A simple model is proposed for the influence of the oxidation state of the soluble enzyme on the distribution of ATPase-inactive and ATPase-active subunit configurations.
叶绿体偶联因子1复合物（CF1）含有一个ε亚基，该亚基可抑制CF1的ATP酶活性。用氯仿处理莱茵衣藻类囊体膜后，仅使明显缺乏δ亚基的酶形式溶解。本文描述了四个相互关联的观察结果。（1）比较了二硫苏糖醇（DTT）诱导的CF1（-δ）的ATP酶激活以及DTT诱导的从CF1（-δ）前体形成物理上可分辨的CF1（-δ，ε）。这两种现象相似的时间进程表明，ε亚基的解离是DTT诱导可溶性CF1的ATP酶激活过程中的一个必要过程。（2）通过可区分的寡聚体之间的亚基交换证明了CF1的ε亚基的可逆解离。将缺乏δ和ε亚基的35S标记的叶绿体偶联因子1 [CF1（-δ，ε）]添加到仅缺乏δ亚基的非放射性偶联因子1 [CF1（-δ）]溶液中。通过高分辨率阴离子交换色谱分离两种酶形式后，在含有CF1（-δ）的色谱级分中检测到放射性。（3）在去除DTT后的几天内，可以将缺乏ε的CF1与经DTT预处理的含ε的CF1分离。另一方面，将经DTT预处理的含ε的CF1与低浓度的邻碘代苯甲酸短暂孵育，得到的色谱图中仅含有含ε的CF1的峰。对这一现象的一个简单解释是，用DTT还原CF1会使ε亚基的表观解离常数从氧化酶的小于或等于5×10-11 M增加到估计的3.5×10-8 M（±1.0×10-8 M）。（4）ATP酶活性数据表明，缺乏ε的酶的氧化并没有完全抑制其明显的活性，但经DTT预处理的含ε亚基的CF1的氧化完全抑制了明显的活性。提出了一个简单的模型，用于解释可溶性酶的氧化状态对ATP酶无活性和ATP酶活性亚基构型分布的影响。
