Cook Gregory M, Keis Stefanie, Morgan Hugh W, von Ballmoos Christoph, Matthey Ulrich, Kaim Georg, Dimroth Peter
Department of Microbiology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.
J Bacteriol. 2003 Aug;185(15):4442-9. doi: 10.1128/JB.185.15.4442-4449.2003.
We describe here purification and biochemical characterization of the F(1)F(o)-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F(1)F(o)-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F(1) subunits alpha, beta, gamma, delta, and epsilon and F(o) subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F(1)F(o) holoenzyme or the isolated F(1) moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F(1). After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Deltapsi). A transmembrane proton gradient or sodium ion gradient in the absence of Deltapsi was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na(+)/H(+) antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na(+) ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.
我们在此描述了从嗜热嗜碱微生物芽孢杆菌属TA2.A1菌株中纯化F(1)F(o)-ATP合酶并对其进行生化特性分析的过程。纯化后的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶上呈现出F(1)F(o)-ATP合酶典型的亚基图谱，包含F(1)亚基α、β、γ、δ和ε以及F(o)亚基a、b和c。这些亚基通过N端蛋白质测序和质谱鉴定。TA2.A1菌株的ATP合酶的一个显著特征是其ATP水解活性受到特异性阻断。使用去污剂月桂基二甲基氧化胺（LDAO）可使ATP酶活性显现出来，LDAO激活ATP水解的倍数超过15倍。这种激活对于F(1)F(o)全酶或分离出的F(1)部分是相同的，因此潜在的ATP水解活性是F(1)的固有特性。重新组装到蛋白脂质体中后，该酶催化由人工诱导的跨膜电势（Δψ）驱动的ATP合成。在没有Δψ的情况下，跨膜质子梯度或钠离子梯度不足以驱动ATP合成。质子载体羰基氰化物间氯苯腙可消除ATP合成，而电中性的Na(+)/H(+)反向转运体莫能菌素则没有影响。Na(+)离子既不刺激ATP合成也不刺激ATP水解，这表明质子是TA2.A1菌株ATP合酶的偶联离子，正如先前针对嗜温嗜碱芽孢杆菌属物种所记录的那样。ATP合酶在其c亚基处被N,N'-二环己基碳二亚胺特异性修饰，这种修饰抑制了ATP合成。
