Kinetic characterization of the ATP-dependent proton pump in bacterial photosynthetic membranes: a study with the fluorescent probe 9-amino-6-chloro-2-methoxyacridine

Add time:07/27/2019    Source:sciencedirect.com

In bacterial photosynthetic membranes the generation of a transmembrane ΔpH coupled to ATP hydrolysis is detected by monitoring the quenching of fluorescence of the monoamine 9-amino-6-chloro-2-methoxyacridine (ACMA), induced in the dark upon addition of the substrates of the ATPase reaction. When Mg-ATP is the reaction complex, kinetic analysis of the hydrolytic and proton pumping activities indicates a similar affinity for the substrate (Km = 0.20 ± 0.05 mM) and maximal rates of ΔpH generation and ATP hydrolysis of 0.37 ± 0.05 ΔpH units s−1 and 10 ± 2 nmol ATP hydrolyzed s−1. (μmol BChl)−1, respectively. A comprehensive investigation of the effects of previously known modulators and/or effectors of the ATPase activity on the proton pump shows that in bacterial photosynthetic membranes, ΔpH generation is strictly regulated in parallel with ATP hydrolysis. These results validate the use of ACMA fluorescence to monitor transmembrane ΔpH of 0.1–1.5 units and to detect F1-F0 functional interactions.

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