Both succinate and NADH caused fluorescence quenching, which was

Both succinate and NADH caused fluorescence quenching, which was eased after the addition of an uncoupler, proving that the observed quenching was indeed caused by the PMF (Fig. 1a). Quenching reached a maximum after ∼10 min (Fig. 1a), significantly slower than IMVs from Escherichia coli under identical conditions (data not shown). This slow quenching may be caused by a larger percentage of leaky IMVs. The lower PMF observed with NADH (11% quenching) compared with succinate (39%) might be due to the partial detachment of the membrane-associated type-II NADH-dehydrogenase

(NDH-II), the main NADH-oxidizing enzyme of the respiratory electron transport chain in M. bovis BCG (Boshoff RG7422 mouse et al., 2004; Weinstein et al., 2005). From these results, it can be concluded that the IMVs are functional. Similarly, IMVs from the fast-growing M. smegmatis accepted both NADH and succinate as electron donors (Fig. 1b). We then investigated whether the IMVs can establish a PMF with ATP as a substrate. No significant quenching was detected either for M. bovis BCG or for M. smegmatis, even after an extended (>30 min) incubation time (Fig. 1a and b). The very small intensity decrease directly after ATP addition is due to sample volume increase and is not reverted with the addition of an uncoupler. Neither variation of the ATP/Mg2+ ratio

(from 0.4 : 1 to 2 : 1), or variation of the pH value (pH 5.5–8.0) nor preparation of IMVs

from M. bovis BCG cultured in an oxygen depletion model (Wayne system) led to detectable quenching upon ATP addition. These results indicate that mycobacterial ATP synthase is not carrying out ATP-hydrolysis-driven proton transport. To exclude the possibility that the observed lack of ATP-hydrolysis-driven Megestrol Acetate proton transport is caused by an extremely low number of ATP synthase molecules in the mycobacterial membrane or by of the detachment of the extrinsic F1 part of ATP synthase, we compared the DCCD-sensitive activities in ATP synthesis and ATP hydrolysis. As shown in Table 1, the IMVs from both M. bovis BCG and M. smegmatis were active in ATP synthesis with specific activities of 0.27 and 0.96 nmol min−1 mg−1, respectively. In contrast, we could not detect any significant DCCD-sensitive ATP hydrolysis activity in IMVs from M. bovis BCG. For M. smegmatis IMVs, DCCD-sensitive ATP hydrolysis activity was detectable, but >4-fold lower as compared with ATP synthesis (Table 1). For an enzyme working with equal speed in both directions, the ATP hydrolysis activity is expected to be higher than the synthesis activity, for example ∼10-fold for ATP synthase from Bacillus PS3 (Bald et al., 1998, 1999). This effect is due to the presence of enzymes in leaky vesicles, unavoidably present in IMV preparations, which can split ATP, but are unable to synthesize it.

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