title: Quantitative and rapid estimation of h fluxes in membrane vesicles : software for analysis of fluorescence quenching and relaxation.
abstract: Proton transport is often visualized in membrane vesicles by use of fluorescent monoamines which accumulate in acidic intravesicular compartments and undergo concentration-dependent fluorescence quenching. Software for an IBM microcomputer is described which permits logging and editing of changes in fluorescence monitored by a Perkin-Elmer LS-5 luminescence spectrometer. An accurate estimate of the instantaneous rate of fluorescence quenching or recovery is then facilitated by least squares fitting of fluorescence data to a nonlinear function. The software is tested with tonoplast vesicles from Beta vulgaris. Quenching of acridine orange fluorescence by ATP-driven (primary) transport and relaxation of quenching by Na(+)/H(+) antiport can both be fitted with single exponential functions. Initial rates of ATP- and Na(+) -dependent fluorescence changes are derived and can be used for K(m) determinations. The method constitutes a simple and efficient alternative to manual analysis of analog fluorescence traces and results in a reliable quantitative measurement of the relative rate of proton transport in membrane vesicle preparations.
authors: Department of Biology, University of York, Heslington, York Y01 5DD, United Kingdom.
authors: I R
authors: IR
authors: Jennings
authors: P A
authors: PA
authors: Rea
authors: R A
authors: RA
authors: Leigh
authors: D
authors: D
authors: Sanders