A Microcomputer-Based System for the Recording and Analysis of Fluorescence Transients from Membrane Vesicles

Jennings, Ian R.; Rea, Philip A.; Sanders, Dale

doi:10.1007/978-1-4684-5341-6_8

Plant Vacuoles 1987

DOI: 10.1007/978-1-4684-5341-6_8

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A Microcomputer-Based System for the Recording and Analysis of Fluorescence Transients from Membrane Vesicles

Ian R. Jennings

Philip A. Rea

Dale Sanders

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1988

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“…Using tonoplast vesicles from Beta vulgaris L. as a model, we show that time-dependent fluorescence change resulting from activation of primary and secondary H+ transport systems can be simply and accurately estimated by least squares fitting of single exponential functions to digitized, stored data. A preliminary report of this work has appeared previously (13).…”

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Quantitative and Rapid Estimation of H⁺ Fluxes in Membrane Vesicles

Jennings¹,

Rea²,

Leigh³

et al. 1988

Plant Physiol.

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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 Proton transport plays a central role in energy transduction in plants (11). Primary H+ transport at the energy-coupling membranes of chloroplasts and mitochondria is driven by light and redox potential energy, respectively, and results in the formation of a transmembrane electrochemical H+ gradient (AdZH+).3 A dissipative flow of protons down this gradient is then coupled to synthesis of ATP. At the plasma membrane and tonoplast, hydrolysis of phosphoanhydride bonds is used to energize H + transport, with reverse flow of H+ down the resulting (A-AH+. wering transport of other solutes through discrete secondary systems.Classical electrophysiological techniques have given insight into the kinetics of H + transport across the plasma membrane of intact plant cells (1) and patch clamp executed in a 'whole cell mode' enables the study of ATP-dependent H + currents in intact vacuoles (4, 12). More commonly, however, H+ transport is studied in isolated membrane vesicles, whose small size prohibits the electrophysiological approach. Further, the fact that 3H rap- ' With the exception of nonsteady state investigations of photosynthetic systems in the ms time range (14), the kinetics of vesicular transmembrane H + translocation have been calculated from analog recordings. Thus, rates have been estimated from hand-drawn tangents to curves-a process which is both laborious and errorsome. The problem is particularly acute for secondary transport where the pH gradient is often generated artificially: because of the intrinsic permeability of the membrane to H+ and the high surface area:volume ratio of vesicles, secondary transport is often initiated against a shifting baseline and the signal obtained is short-lived owing to depletion of the limited reserves of intravesicular protons.Here we report a method for the logging and subsequent analysis of fluorescence data from ApH-reporting probes in membrane vesicles. Using tonoplast vesicles from Beta vulgaris L. as a model, we show that time-dependent fluorescence change resulting from activation of primary and secondary H+ transport systems can be simply and accurately estimated by least squares fitting of single exponential functions to digitized, stored data. A preliminary report of this work has appeared previously (13). MATERIALS AND METHODS

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confidence: 99%

Quantitative and Rapid Estimation of H⁺ Fluxes in Membrane Vesicles

Jennings¹,

Rea²,

Leigh³

et al. 1988

Plant Physiol.

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A Microcomputer-Based System for the Recording and Analysis of Fluorescence Transients from Membrane Vesicles

Cited by 1 publication

References 1 publication

Quantitative and Rapid Estimation of H⁺ Fluxes in Membrane Vesicles

Quantitative and Rapid Estimation of H⁺ Fluxes in Membrane Vesicles

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A Microcomputer-Based System for the Recording and Analysis of Fluorescence Transients from Membrane Vesicles

Cited by 1 publication

References 1 publication

Quantitative and Rapid Estimation of H+ Fluxes in Membrane Vesicles

Quantitative and Rapid Estimation of H+ Fluxes in Membrane Vesicles

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Quantitative and Rapid Estimation of H⁺ Fluxes in Membrane Vesicles

Quantitative and Rapid Estimation of H⁺ Fluxes in Membrane Vesicles