Orientation of the protonmotive force in membrane vesicles of escherichia coli

✍ Scribed by Adler, Lawrence W. ;Ichikawa, Tomio ;Hasan, Syed M. ;Tsuchiya, Tomofusa ;Rosen, Barry P.

• Publisher: Wiley (John Wiley & Sons)
• Year: 1977
• Tongue: English
• Weight: 719 KB
• Volume: 7
• Category: Article
• ISSN: 0091-7419
• DOI: 10.1002/jss.400070103

✦ Synopsis

Abstract

Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D‐lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside.

The latter type of vesicle, on the other hand, exhibits D‐lactate‐dependent proline transport but does not accumulate calcium with D‐lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D‐lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy‐linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).