The Increase in the Number of Accessible SH-Groups in the Enterococcal Membrane Vesicles by ATP and Nicotinamide Adenine Dinucleotides Anna Poladyan, Armen Trchounian Department of Biophysics of the Biological Faculty, Yerevan State University, 1 Alex Manoukian Str., 375025 Yerevan, Armenia Received: 10 September 2005 / Accepted: 3 November 2005 Abstract. The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD + +NADH) to the vesicles caused a 4-fold or 1.9-fold increase in the number of SH-groups, respectively. This was inhibited by treatment with N-ethylma- leimide. The increase was significant when ATP and NAD + +NADH both were added. The change was lacking in the presence of the F 0 F 1 -ATPase inhibitors N,N¢-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F 0 F 1 -ATPase and, in addition, it was less in potassium ion–free medium. These results are correlated with data about K + -dependent F 0 F 1 -ATPase activity, suggesting a relationship between the F 0 F 1 -ATPase and K + uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes. Many proteins are known to contain thiol-groups in the form of cysteine residues, the oxidation-reduction states of which can modulate the enzymatic and transport activity of bacteria and the other cells (for reviews, see [7, 19]). In Escherichia coli, the number of accessible SH- groups in membrane vesicles has been shown recently by Mnatsakanyan et al. [14] to be increased by ATP or by formate, suggesting an interaction between the F 0 F 1 - ATPase and hydrogenase 4 or hydrogenase 3, compo- nents of formate hydrogenlyases, under fermentation conditions. This would lead to formation of a protein– protein complex [1,2] within which the energy could be transferred via a dithiol-disulfide interchange [11]. The latter is likely to be an interesting energy-transferring mechanism among different bacteria in fermentation conditions. Under fermentation, the Enterococcus hirae H + - translocating ATP synthase (F 0 F 1 )-ATPase operates as an ATP-driven H + pump to generate proton motive force (Dl H + ) [8]. The latter mediates many energy-dependent processes and modulates the enzymatic activity of the membrane. However, F 0 F 1 might have direct involve- ment with secondary solute transport systems such as K + uptake Trk-like or KtrI system. The findings about a fixed stoichiometry of K + influx via Trk-like system with H + efflux through the F 0 F 1 -ATPase [12, 22] and the F 0 F 1 -ATPase activity strongly stimulated by K + [22] seem to be serious arguments supporting the idea that the F 0 F 1 -ATPase is functioning in a closed relationship with Trk-like system. The energy of ATP is proposed to be transferred from F 0 F 1 to Trk-like system through a dithiol-disulfide interchange. In this regard, we expect that addition of ATP may cause a change in accessible SH-groups. This article presents data showing that, in mem- brane vesicles from E. hirae grown at alkaline condi- tions, the number of accessible SH-groups is increased by ATP and by nicotinamide adenine dinucleotides. These effects might be related to the F 0 F 1 -ATPase and largely dependent on the presence of K + . Materials and Methods Bacterial strains and growth, membrane vesicles. The wild-type strain E. hirae ATCC9790 and the atpD mutant strain MS116 (defective in the b subunit of F 1 ) [22] were used in this study. The Correspondence to: A. Trchounian; email: Trchounian@ysu.am CURRENT MICROBIOLOGY Vol. 52 (2006), pp. 300–304 DOI: 10.1007/s00284-005-0293-6 Current Microbiology An International Journal ª Springer Science+Business Media, Inc. 2006