Schlegel Katharina, Müller Volker
Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Goethe-University Frankfurt, Frankfurt, Germany.
Methods Enzymol. 2011;494:233-55. doi: 10.1016/B978-0-12-385112-3.00012-3.
Methanogens are the only significant biological producers of methane. A limited number of C(1) substrates, such as methanol, methylamines, methyl sulfate, formate, H(2)+CO(2) or CO, and acetate, serve as carbon and energy source. During degradation of these compounds, a primary proton as well as a primary sodium ion gradient is established, which is a unique feature of methanogens. This raises the question about the coupling ion for ATP synthesis by the unique A(1)A(o) ATP synthase. Here, we describe how to analyze and determine the Na(+) dependence of two model methanogens, the hydrogenotrophic Methanothermobacter thermautotrophicus and the methylotrophic Methanosarcina barkeri. Furthermore, the determination of important bioenergetic parameters like the ΔpH, ΔΨ, or the intracellular volume in M. barkeri is described. For the analyses of the A(1)A(O) ATP synthase, methods for measurement of ATP synthesis as well as ATP hydrolysis in Methanosarcina mazei Gö1 are described.
产甲烷菌是甲烷唯一重要的生物生产者。少数几种C(1)底物，如甲醇、甲胺、硫酸甲酯、甲酸、H(2)+CO(2)或CO以及乙酸盐，可作为碳源和能源。在这些化合物的降解过程中，会建立起一个初级质子梯度以及一个初级钠离子梯度，这是产甲烷菌的一个独特特征。这就引发了关于独特的A(1)A(o)ATP合酶合成ATP时的偶联离子的问题。在此，我们描述了如何分析和确定两种模式产甲烷菌——嗜氢自养型嗜热栖热甲烷杆菌和甲基营养型巴氏甲烷八叠球菌对Na(+)的依赖性。此外，还描述了在巴氏甲烷八叠球菌中测定重要生物能量学参数（如ΔpH、ΔΨ或细胞内体积）的方法。对于A(1)A(O)ATP合酶的分析，描述了在马氏甲烷球菌Gö1中测量ATP合成以及ATP水解的方法。
