Hisabori T, Kato Y, Motohashi K, Kroth-Pancic P, Strotmann H, Amano T
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Yokohama, Japan.
Eur J Biochem. 1997 Aug 1;247(3):1158-65. doi: 10.1111/j.1432-1033.1997.01158.x.
The expression plasmids for the subunit gamma (gamma(c)) and the subunit epsilon (epsilon(c)) of chloroplast coupling factor (CF1) from spinach were constructed, and the desired proteins were expressed in Escherichia coli. Both expressed subunits were obtained as inclusion bodies. When recombinant gamma(c) was mixed with recombinant alpha and beta subunits of F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (alpha3beta3gamma(c)) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCl2, which induces formation of a disulfide bond between two cysteine residues in gamma(c). Hence, this complex has similar modulation characteristics as CF1. The effects of recombinant epsilon(c) and epsilon subunit from TF1 (epsilon(t)) on alpha3beta3gamma(c) were also investigated. Epsilon(c) strongly inhibited the ATP hydrolysis activity of chimeric alpha3beta3gamma(c) complex but epsilon(t) did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium. The addition of epsilon(c) or epsilon(t) to the alpha3beta3gamma(t) complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity. These results suggest that (a) the specific regulatory function of gamma(c) can be transferred to the bacterial subunit complex; (b) the interaction between the gamma(c) subunit and epsilon(c) strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the alpha3beta3 core.
构建了菠菜叶绿体偶联因子（CF1）亚基γ（γ(c)）和亚基ε（ε(c)）的表达质粒，并在大肠杆菌中表达了所需蛋白质。两个表达的亚基均以包涵体形式获得。当重组γ(c)与嗜热芽孢杆菌PS3（TF1）的F1重组α和β亚基混合时，重建了嵌合亚基复合物（α3β3γ(c)），并显示出显著的ATP水解活性。在二硫苏糖醇存在下，该复合物的ATP水解活性增强，而添加CuCl2则抑制其活性，CuCl2可诱导γ(c)中两个半胱氨酸残基之间形成二硫键。因此，该复合物具有与CF1相似的调节特性。还研究了重组ε(c)和TF1的ε亚基（ε(t)）对α3β3γ(c)的影响。ε(c)强烈抑制嵌合α3β3γ(c)复合物的ATP水解活性，但ε(t)没有。当向测定培养基中加入甲醇时，抑制作用消除，ATP水解活性恢复。向TF1的真实亚基复合物α3β3γ(t)中添加ε(c)或ε(t)，会导致ATP水解活性受到微弱刺激。这些结果表明：（a）γ(c)的特定调节功能可以转移到细菌亚基复合物中；（b）γ(c)亚基与ε(c)之间的相互作用强烈影响酶活性，该酶活性在位于α3β3核心的催化位点上催化。
