Effect of Inhibitors on the Substrate‐Dependent Quenching of 9‐Aminoacridine Fluorescence in Inside‐Out Membrane Vesicles of Escherichia coli

Abstract

The effect of various inhibitors on the substrate-dependent fluorescence quenching of 9-aminoacridine was measured in inside-out membrane vesicles of E. coli. The fluorescence quenching rate in the presence of inhibitors was dependent on the electron transfer rate through the respiratory chain with NADH, succinate, D-lactate or DL-glycerol 3-phosphate as substrates. Several patterns of response were given by the inhibitors. Inhibitors competitive with substrate or those acting only on the dehydrogenases gave a direct relationship between the extent of oxidase activity inhibition and the rate of quenching. A biphasic relationship was given by 2-heptyl-4-hydroxyquinoline N-oxide and piericidin A which was due to these compounds acting as inhibitors of the respiratory chain and, at higher concentrations, as uncoupling agents. Uncouplers inhibited fluorescence quenching with minimal inhibition of oxidase activity. The transmembrane pH difference was calculated from the extent of fluorescence quenching and the intravesicular volume. The maximum pH difference of 3.3-3.7 units was generated by each of the substrates tested.