Mechanisms of Active Transport in Isolated Bacterial Membrane Vesicles: VII. FLUORESCENCE OF 1-ANILINO-8-NAPHTHALENESULFONATE DURING d-LACTATE OXIDATION BY MEMBRANE VESICLES FROM ESCHERICHIA COLI

Abstract

Abstract Addition of d-lactate to Escherichia coli membrane vesicles results in a rapid decrease in the fluorescence of 1-anilino-8-naphthalenesulfonate; the decreased level of fluorescence is maintained until the reaction mixture becomes anaerobic, at which time there is a large increase in fluorescence. The rate of the initial fluorescence decrease is most marked with d-lactate; it is much slower with l-lactate, dl-α-hydroxybutyrate, succinate, and reduced diphosphopyridine nucleotide. ATP has no effect. Potassium cyanide and 2-heptyl-4-hydroxyquinoline-N-oxide, which inhibit electron transfer after the site of energy coupling, reverse the initial decrease in fluorescence. Oxamic acid, p-chloromercuribenzoate, and N-ethylmaleimide, which inhibit before or at the site of energy coupling, cause a further decrease in fluorescence. Dithiothreitol reverses the effect of p-chloromercuribenzoate. Each of these reagents, as well as the uncoupling agent carbonyl cyanide-m-chlorophenylhydrazone, markedly inhibit the rate of the initial fluorescence decrease induced by d-lactate. These effects correlate nearly perfectly with the behavior of the d-lactic dehydrogenase-coupled transport systems described previously. Membrane vesicles treated with mouse duodenal phospholipase or extracted with acetone exhibit similar fluorescence changes on addition of d-lactate. Moreover, each of the fluorescence changes described appears to be identical, whether the dye is excited directly or by energy transfer from the membrane proteins. These results indicate that the fluorescence changes observed reflect structural transitions in components of the membrane.