In vitro activity: ETH2120 (also known as Sodium ionophore III) is a Na+ ionophore that is suitable for the assay of sodium activity in blood, plasma, serum. etc.The sodium ionophore ETH2120, but not protonophores, stimulated hydrogen-dependent caffeate reduction by 280%, indicating that caffeate reduction is coupled to the buildup of a membrane potential generated by primary Na(+) extrusion. Caffeate reduction was coupled to the synthesis of ATP, and again, ATP synthesis coupled to hydrogen-dependent caffeate reduction was strictly Na(+) dependent and abolished by ETH2120, but not by protonophores, indicating the involvement of a transmembrane Na(+) gradient in ATP synthesis. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) abolished ATP synthesis, and at the same time, hydrogen-dependent caffeate reduction was inhibited. This inhibition could be relieved by ETH2120. These experiments are fully compatible with a chemiosmotic mechanism of ATP synthesis with Na(+) as the coupling ion during hydrogen-dependent caffeate reduction by A. woodii.

Kinase Assay:

Cell Assay: ells were grown up to an A600 of 0.15 to 0.25. Then, caffeate was added from an 0.1 M stock solution to induce the cells' ability to reduce caffeate. Cultures were harvested anaerobically at the end of the exponential growth phase by centrifugation (2,700 × g, 10 min, 4°C) and washed three times with imidazole-HCl buffer (20 mM imidazole-HCl, 20 mM MgSO4, 5 mM dithioerythritol, 1 mg of resazurin per liter, pH 7). The cells were resuspended in the same buffer to a final protein concentration of 11 to 16 mg/ml under an atmosphere of N2-H2 (95:5 [vol/vol]). This suspension was stored on ice and used immediately for the experiments. The protein concentration of the cell suspension was determined as described previously. All manipulations were done under strictly anaerobic conditions in an anaerobic chamber