Pisa Kim Y, Huber Harald, Thomm Michael, Müller Volker
Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe Universität Frankfurt, Frankfurt, Germany.
FEBS J. 2007 Aug;274(15):3928-38. doi: 10.1111/j.1742-4658.2007.05925.x. Epub 2007 Jul 5.
The rotor subunit c of the A(1)A(O) ATP synthase of the hyperthermophilic archaeon Pyrococcus furiosus contains a conserved Na(+)-binding motif, indicating that Na(+) is a coupling ion. To experimentally address the nature of the coupling ion, we isolated the enzyme by detergent solubilization from native membranes followed by chromatographic separation techniques. The entire membrane-embedded motor domain was present in the preparation. The rotor subunit c was found to form an SDS-resistant oligomer. Under the conditions tested, the enzyme had maximal activity at 100 degrees C, had a rather broad pH optimum between pH 5.5 and 8.0, and was inhibited by diethystilbestrol and derivatives thereof. ATP hydrolysis was strictly dependent on Na(+), with a K(m) of 0.6 mM. Li(+), but not K(+), could substitute for Na(+). The Na(+) dependence was less pronounced at higher proton concentrations, indicating competition between Na(+) and H(+) for a common binding site. Moreover, inhibition of the ATPase by N',N'-dicyclohexylcarbodiimide could be relieved by Na(+). Taken together, these data demonstrate the use of Na(+) as coupling ion for the A(1)A(O) ATP synthase of Pyrococcus furiosus, the first Na(+) A(1)A(O) ATP synthase described.
嗜热古菌激烈火球菌的A(1)A(O) ATP合酶的转子亚基c含有一个保守的Na(+)结合基序，这表明Na(+)是一种偶联离子。为了通过实验确定偶联离子的性质，我们通过用去污剂从天然膜中增溶，然后采用色谱分离技术来分离该酶。制备物中存在整个膜嵌入的马达结构域。发现转子亚基c形成一种抗SDS的寡聚体。在所测试的条件下，该酶在100℃时具有最大活性，在pH 5.5至8.0之间有相当宽的最适pH值，并且受到己烯雌酚及其衍生物的抑制。ATP水解严格依赖于Na(+)，K(m)为0.6 mM。Li(+)而非K(+)可以替代Na(+)。在较高质子浓度下，Na(+)依赖性不太明显，表明Na(+)和H(+)在一个共同结合位点上存在竞争。此外，N',N'-二环己基碳二亚胺对ATP酶的抑制作用可被Na(+)缓解。综上所述，这些数据证明了Na(+)作为激烈火球菌A(1)A(O) ATP合酶的偶联离子的作用，这是所描述的首个Na(+) A(1)A(O) ATP合酶。
