Energetics of active transport in inverted membrane vesicles of Escherichia coli

Abstract

Inside-out (ISO) membrane vesicles isolated from Escherichia coli are inverted (interior positive and acidic) with respect to right-side-out (RSO) membranes (interior negative and alkaline). As the membranes are coupled with an electron donor (D-lactate) as the energy source, ISO membranes exhibit a proton gradient (ΔpH) of 46.4 mV and an electrical potential (ΔΨ) of 63.3 mV, as measured by a flow dialysis method. This gives a proton electrochemical potential (Δ μ H+) of 110 mV across the membranes. The energetics of the formation of Δ μ H+ across ISO membranes were investigated by microcalorimetry in combination with oxygen uptake measurement. The results were compared with those previously obtained with RSO membranes. In these two types of membranes, the energetic parameters in the formation of Δ μ H+ (the enthalpy (Δ H m), the free energy (Δ G m) and the entropy (Δ S m) changes) exhibit similar positive signs. The observations suggest that the characteristics of the created Δ μ H+ across these membranes are analogous, regardless of the polarities across the membranes. The efficiency in the free energy transfer from the energy source (D-lactate oxidation reaction) to the creation of Δ μ H+ is essentially the same in ISO and RSO membranes, while the enthalpy transfer is much lower in ISO than in RSO membranes. The discrepancy in the enthalphy transfer is associated with the intrinsic difference between ISO and RSO membranes in the direction of proton movement. The employed thermochemical method can not only confirm the formation of Δ μ H+ across ISO membranes, but also reveals a significant difference in the value of ΔH m between ISO and RSO membranes, due to an opposite polarity across the membranes. This work provides an understanding of the role that membrane polarity plays in the energetics of active transport across membranes.