Abstract
Facilitated diffusion of [14C]lactose into inverted membrane vesicles of Escherichia coli was measured using HgCl2 as a stopping reagent and polylysine to flocculate the vesicles for filtration. Equilibration of lactose between the internal and external volumes required expression of the y gene of the lac operon and was inhibited by thiodigalactoside or by prior incubation with N-ethylmaleimde or HgCl2. The initial rate of uptake was saturable, with a Kt of 0.95 mM. Counterflow of [14C]lactose was demonstrated in either direction. ATP hydrolysis or respiration drove the efflux of internal lactose. The effect of ATP required addition of F1 coupling factor (ATPase) from E. coli when lactose transport was studied in F1-deficient inverted vesicles. Accumulation of lactose against a concentration gradient was achieved by forming an artificial electrochemical proton gradient consisting of a membrane potential negative inside or a pH gradient basic inside. Addition of ATP inhibited this proton driven uptake showing that it occurred in inverted vesicles. It was concluded that the lactose-proton co-transport protein (M protein) is qualitatively symmetrical with respect to the facilitated diffusion of lactose and the coupling of proton and lactose transport.
Highlights
Facilitated diffusion of [‘“C llactose into inverted mem- chemical proton gradient, the magnitude of which correlates brane vesicles of Escherichia coli was measured using HgCI, well with the abilities of the different substrates to stimulate as a stopping reagent and polylysine to flocculate the transport [6]
Inverted membrane vesicles from E. coli take up [‘“Cllactose in a time-dependent manner
The transport occurs in the absence of respiration or ATP hydrolysis and is uncoupler insensitive showing that it is facilitated diffusion, which has not been reported previously for isolated membrane preparations
Summary
Facilitated diffusion of [‘“C llactose into inverted mem- chemical proton gradient, the magnitude of which correlates brane vesicles of Escherichia coli was measured using HgCI, well with the abilities of the different substrates to stimulate as a stopping reagent and polylysine to flocculate the transport [6]. Kaback et al [4, 5] have shown that addition of respiratory substrate to a preparation of isolated membrane vesicles results in the accumulation of gradient and the transporter is capable of coupling galactoside to proton flux in either direction. If this is true, reversing the proton gradient should result in a reversal of galactoside transport. We report here studies of lactose transport by these inverted vesicles, which demonstrate facilitated diffusion and symmetrical coupling of lactose transport to the electrochemical proton gradient
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