Jean-Pierre Grouzis

Quantitative measurement of cationic fluxes, selectivity and membrane potential using liposomes multilabelled with fluorescent probes.

Liposomes of egg PC/PG (8:2, mol/mol) were multilabelled with PBFI, pyranine and oxonol VI, fluorescent probes for, respectively, K+, H+ and membrane potential. Monitoring fluorescence with a multichannel photoncounting spectrofluorometer during K+ filling experiments allowed to measure K+ influx, the associated H+ efflux and the membrane potential, continuously and simultaneously. The proton net efflux quantitatively mirrored the K+ net influx. The rate of the K+/H+ exchange diminished progressively as a quasi-equilibrium was reached for both K+ and H+. In the presence of valinomycin, the measured membrane potential during the K+ filling actually corresponded to the Nernst potential calculated from the observed K+ gradient. In the absence of valinomycin, it corresponded to the Nernst potential calculated from the observed H+ gradient. In the latter case, the permeability coefficient of liposomes to K+, calculated from the Goldman-Hodgkin-Katz relation, was 6.10(-13) m s-1. The selectivity sequence for alkali cations of liposomes was determined from the measured H+ efflux associated to the influx of the different cations. The selectivity sequence corresponded to the series VI of Eisenman, suggesting interaction of the cation with an anionic field of intermediate strength.

Electrostatic characteristics of corn root plasmalemma: effect on the Mg2+-ATPase activity

The macroscopic surface potential of corn root plasmalemma was determined from the microelectrophoretic mobility of the membrane vesicles. It was compared to a microscopic surface potential obtained from the binding of the ANS probe on its lipidic site. These two potentials are in good agreement whatever the used ionic strengths. Various univalent salts were shown to depolarize the membrane surface via the aspecific screening effect which was predicted by the Gouy-Chapman model. The addition of 50 mM KCl increased the apparent affinity of the ATPase for MgATP. It was assumed that the electrostatical repulsion of MgATP (valency = −1.5 at pH 6.5) was responsible for the difference between the apparent Km and the intrinsic one. The macroscopic surface potential values were used to calculate the intrinsic Km of the enzyme and led to the same value whatever the univalent salt concentration was. Another method of calculation of the intrinsic Km was used, which did not rely on surface potential measurements. The intrinsic Km values calculated by this way had the same value as the ones estimated from the experimental determinations of the surface potential. This result implies that the microscopic surface potential in the vicinity of the ATPase catalytic site has the same value as the two other potentials. Membrane proteins were extracted and adsorbed on latex beads, which had the same electrophoretic mobility as the plasmalemma vesicles. The proteic and lipidic charges thus create an electrostatic surface which appears uniformly smeared in the presence of univalent salts. In the presence of MgCl2, both the surface potential of the protein-coated beads and the potential sensed by MgATP near its catalytic site on the vesicles were zero. Nevertheless, the macroscopic surface potential of the vesicles remained negative. These results pointed out the heterogeneity of the surface charge density which was induced by Mg2+. They suggest that the uniformity which was observed in the absence of divalent ion was not the result of a cancellation of local heterogeneities by averaging effects. This uniformity seems to result from identical local charge densities above the lipidic and proteic parts of the membrane.

Electrophysiological Study with Oxonol VI of Passive NO3− Transport by Isolated Plant Root Plasma Membrane

In contrast to animal cells, plant cells contain approximately 5-50 mM nitrate in cytosol and vacuole. The lack of specific spectroscopic probes, or suitable isotopes, impedes in vitro studies of NO3- transport. Reconstitution of root cell plasma membrane (PM) proteins in mixed soybean lipid:egg phosphatidylcholine allowed for the generation of large K+-valinomycin diffusion potentials (Em), monitored with the oxonol VI dye. Nevertheless, Em was restricted to approximately 130 mV by capacitor properties of biological membranes. This caused an increasing discrepancy at higher K+-Nernst potentials used for calibration. Therefore, Em was determined directly from the fluorescence of the dye free in buffer, bound at zero Em, and bound upon Em generation. Then, an electrophysiological analysis of the NO3--dependent dissipation rate of Em gave the net passive flux (JN) and the permeability coefficient to NO3- (PN). The plant root cell PM exhibited a strikingly large PN (higher than 10(-9) m s-1) at high Em (90-100 mV) and pH 6.5. At low Em (50-60 mV) and pH 7.4, PN decreased by 70-fold and became similar to that of the lipid bilayer. This agreed with the previous observation that 15 mM NO3- short-circuits the plant root PM H+-ATPase at its optimal pH of 6.5.

In vitro study of passive nitrate transport by native and reconstituted plasma membrane vesicles from corn root cells

Proteins from phase-partitioned corn root plasma membrane were reconstituted into soybean lipids/egg PC (8:2, w:w) using deoxycholate and rapid gel filtration to eliminate the detergent. All (H+)ATPase molecules were inside-out reinserted and the initial activity was totally recovered in an homogeneous vesicle preparation. In addition, membrane tightness greatly increased, as shown by the size and stability of the response of the fluorescent membrane potential probe (oxonol VI) to an imposed K+ diffusion gradient. Consequently, the H(+)-pumping activity of the (H+)ATPase, monitored with the fluorescent pH probe (ACMA), increased 20-fold after reconstitution. A protein-mediated passive transport of nitrate was first demonstrated by the ability of NO3- to electrically short-circuit the (H+)ATPase in plasma membrane vesicles and not in liposomes containing only the purified enzyme. The passive transport was saturable (K(m) approximately 5 mM), thermolabile, inhibited by the arginine reagent phenylglyoxal, and selective (NO3- > I- approximately ClO3- approximately Br- > Cl- approximately NO2- > Iminodiacetate approximately SO4(2-)). Passive NO3- transport was also determined, independently of the (H+)ATPase, from the NO3(-)-dependent augmentation of the dissipation rate of imposed diffusion potentials. This second transport assay gave similar K(m) for NO3- and should be suitable to continue the functional and biochemical characterization of the NO3- transport system.

Local ionic environment of plant membranes: effects on membrane functions

Summary Electrostatic properties of the isolated plasma membrane of maize roots were studied using the fluorescent ANS probe and microelectrophoresis of membrane vesicles. Variations of the net surface charge of membrane were obtained by adding ionic surfactants or by acidifying the medium. Parallel experiments were performed on excised roots. We showed that surface electrostatic interactions can affect major membrane functions such as ATPase activity, trans-membrane PD, and permeability to NO 3 .

Evidence for the contribution of surface potential to the transtonoplast potential difference measured on isolated vacuoles with microelectrodes

Summary In order to elucidate the origin of the transtonoplast potential difference (PD) measured on isolated vacuoles, the possible contribution of the surface potential to the transmembrane PD was studied. The PD and the electrophoretic mobility were measured on vacuolar preparations, isolated from Beta vulgaris roots and Acer pseudoplatanus cells. Zeta potential was calculated from the electrophoretic mobility. The changes in zeta potential, in response to pH or to a cationic surfactant, were shown to induce parallel variations in the PD. These results suggest that the surface potential could contribute to the transmembrane PD of isolated vacuoles. Various hypotheses on the origin of this transtonoplast PD are discussed.

A test for screening monoclonal antibodies to membrane proteins based on their ability to inhibit protein reconstitution into vesicles

The hypothesis that the binding of an antibody to a membrane protein is likely to prevent the reconstitution of the protein into liposomes was checked, by using the plant plasma membrane H(+)-ATPase (EC 3.6.1.35) as a model system, and two reconstitution procedures: spontaneous insertion (SI) of purified H(+)-ATPase into preformed liposomes, and a detergent-mediated reconstitution (DMR) procedure allowing the reconstitution of the whole membrane protein content. Nine monoclonal antibodies (MABs) raised against H(+)-ATPase were tested. None affected the functioning of the enzyme reconstituted in liposomes, suggesting that the probability to obtain an inhibitory MAB is low. Five MABs inhibited its SI, and seven inhibited its reconstitution in the DMR procedure. These results indicate that it is possible to screen antibodies directed against membrane protein, by making use of their ability to inhibit the reconstitution of these proteins.