Fig 1.
Structures of thymoquinone (TQ).
Fig 2.
Immunoblotting of wild-type purified F1 and membrane bound F1Fo ATP synthase with anti-F1-α antibody.
Wild-type purified F1 (0.4μg) and two membrane bound F1Fo preparations (4μg) were run on 10% SDS-polyacrylamide gel with membranes from null mutants DK8 and pUC118/DK8 controls. Protein bands were transferred to nitrocellulose and immunoblotted using anti-F1-α antibody.
Fig 3.
Complete inhibition of ATPase activity of membrane-bound ATP synthase by TQ.
Membranes were preincubated for 60 min at 23°C with varied concentration of TQ and then 1 ml of ATPase cocktail was added and activity measured. For details are given in Materials and Methods section. Each data point represents average of four experiments done in duplicate tubes, using two independent membrane F1Fo preparations. Thus, mean given with standard error for each inhibitory concentration is N4 where N represents the sample size.
Fig 4.
Reversal of TQ induced inhibition by dilution and passing through centrifuge columns.
Membrane bound ATP synthase (Mbr) or purified F1 (F1) was inhibited with inhibitory concentration of TQ shown in the figure for 60 min under conditions as described in Fig 2. (A), TrisSO4 pH 8.0 buffer was added to bring back the TQ concentration to non-inhibitory level and activity was measured. (B) Purified F1 was incubated with inhibitory concentrations of TQ for 60 min under conditions as described in Fig 3. Then the inhibited samples were passed twice through 1 ml centrifuge columns and ATPase activity was measured.
Table 1.
Thymoquinone (TQ) induced growth inhibition of Escherichia coli cells at 150 μM concentration.