Proton efflux

V. Romheld and D. Kramer, Relationship between proton efflux and rhizodermal transfer eells indueed by iron defieieney. Z Pflanzenphysiol II3 Ti (1983). 

Current estimates are that three protons move into the matrix through the ATP-synthase for each ATP that is synthesized. We see below that one additional proton enters the mitochondrion in connection with the uptake of ADP and Pi and export of ATP, giving a total of four protons per ATP. How does this stoichiometry relate to the P-to-O ratio When mitochondria respire and form ATP at a constant rate, protons must return to the matrix at a rate that just balances the proton efflux driven by the electron-transport reactions. Suppose that 10 protons are pumped out for each pair of electrons that traverse the respiratory chain from NADH to 02, and 4 protons move back in for each ATP molecule that is synthesized. Because the rates of proton efflux and influx must balance, 2.5 molecules of ATP (10/4) should be formed for each pair of electrons that go to 02. The P-to-O ratio thus is given by the ratio of the proton stoichiometries. If oxidation of succinate extrudes six protons per pair of electrons, the P-to-O ratio for this substrate is 6/4, or 1.5. These ratios agree with the measured P-to-O ratios for the two substrates. 

More specifically, compounds like podolactone A (Fig. 10.1) inhibit proton efflux from plant cells induced by fusicoccin, without affecting ATP levels.42 The related compound, podolactone E is a strong inhibitor of 6-aminolevulinic acid and chlorophyll synthesis.34 The authors concluded that this was caused by suppression of synthesis of proteins needed in the porphyrin pathway because podolactones also inhibited gibberellic acid-induced a-amylase synthesis in barley embryos. The molecular target site(s) of this class of terpenoid phytotoxins remains to be determined. 

Sasse, J. M., Cerana, R., and Colombo, R. 1984. The effects of podolactone-type inhibitors on fuscicoccin-induced growth and proton efflux. Physiol. Plant. 62, 303-308... 

The redox system does not depend on endosomal acidification but needs TfR. Fe2Tf first binds to TfR which is located in close proximity to the proton-and electron-pumping NADHiTf oxidoreductase. The Fe—Tf bond is destabilized by proton efflux, making Fe3+ susceptible to reduction. Fe2+ is trapped by a plasma membrane binder and can be transported by a translocator [4]. As Al is a simple trivalent cation incapable of redox changes, it may be theoretically impossible that Al bound to Tf is taken up by a redox mechanism. Actually, no reports on a redox-mediated process of Al bound to Tf have been made. 

Fig. 5.1. ATPase (FqF,) and proton motive ATP synthesis. FqF, reconstituted into liposomes transform energy of proton efflux driven by an electrochemical potential difference of protons across the membrane = FAi — 2.3/ 7 ln ApH) [3,38). F(, (right hand side of the liposome) without F, is an...

HPTS is a pH-sensitive fluorophore (pk, 7.3) [6]. The opposite pH sensitivity of the two excitation maxima permits the ratiometric (i.e. unambiguous) detection of pH changes in double-channel fluorescence measurements. The activity of synthetic ion channels is determined in the HPTS assay by following the collapse of an applied pH gradient. In response to an external base pulse, a synthetic ion channel can accelerate intravesicular pH increase by facilitating either proton efflux or OH influx (Fig. 11.5c). These transmembrane charge translocations require compensation by either cation influx for proton efflux or anion efflux for OH influx, i.e. cation or anion antiport (Fig. 11.5a). Unidirectional ion parr movement is osmotically disfavored (i.e. OH /M or X /H symport). HPTS efflux is possible with pores only (compare Fig. 11.5b/c). Modified HPTS assays to detect endovesiculation (Fig. 11.1c) [16], artificial photosynthesis [17] and catalysis by pores [18] exist. 

More difficult in BLMs, refined HPTS assays exist to address the special cases of selective transport of protons [11] and electrons [17] in LUVs. In the conventional HPTS assay (Fig. 11.5c), the apparent activity of proton channels decreases with increasing proton selectivity because the rate ofthe disfavored cation (M ) influx influences the detected velocity more than the favored proton efflux. Disfavored potassium influx can, however, be accelerated with the potassium carrier vaiinomycin (Fig. 11.8). Increasing activity in the presence of vaiinomycin identifies proton channels with H > K+ selectivity being at least as high as the maximal measurable increase (in unpolarized LUVs of course, compare Section 11.3.4). Important controls include evidence for low enough vaiinomycin concentrations to exclude activity without the proton channel (due to disfavored H+ efflux). The proton carrier FCCP is often used as complementary additive to confirm M+ > H+ selectivity (e.g. amphotericin B). 

The notion that Xg represents a proton gradient created by proton translocation across a closed membrane system (vesicle) was subsedquently demonstrated by Izawa s elegant experiments that show parallel kinetic profiles for Xg build-up as represented by ATP formation and for proton efflux measured directly with a pH-meter, as shown in Fig. 11 (B). 

Fig. 11. A) Kinetics of Xg formation (solid iines) and decay (dashed iines) in chloroplasts at three different pHs during iliumination pH 8.0 was used for the dark-reaction stage. Illumination time is (l in seconds, shown in the bottom scale, and the dark interval (Ay between illumination and rapid mixing of the chloroplast sample with the ADP+Pi+Mg reaction mixture Afo, in seconds, is also shown in the bottom scale. (B) Correspondence of proton efflux and decay of Xg. See text for details. Figure source (A) Hind and Jagendorf (1963) Separation ofiighi and dark stages in photophosphoryiation. Proc Nat Acad Sci, USA 49 719 (B) Izawa (1970) The relationship of post-illumination ATP formation capacity Xg) to H accumulation in chloroplasts. Biochim Biophys Acta 223 168.

Figure II. Formation of a trans-thylakoid proton gradient by intact chloro-plasts as indicated by the quenching of 9-aminoacnaine fluorescence (86, 87), and a subsequent efflux of protons from the thylakoids on darkening. Note accelerated proton efflux in the presence of sodium caprylate. Conditions Intact spinach chloroplasts were suspended in isotonic sorbitol buffer (20 /xg chlorophyll ml 1) ana illuminated with saturating red light in the presence of 0.5 mM methylviologen as electron acceptor.

The membrane potential depolarization in pmal mutants could be explained if the mutant enzymes were less active in pumping protons across the membrane. This notion was supported by kinetic studies on ATP hydrolysis by these enzymes that showed small but significant decreases in Vmax (15). However, a dilemma arose when whole cell medium acidification experiments were performed, which reflect the action of the H+-ATPase in vivo. The rate of glucose-induced proton efflux by pmal mutant cells was found to be considerably better than that of wild-type cells (Figure 2). Only when high external K+ was included in the medium to minimize differences in membrane potential between mutant and wild-type cells did the activity of the... 

The higher the proton motive force, the larger the proton gradient, so the slower the rate of proton efflux from the matrix. Consequently, a high proton gradient slows the flux of electrons along the electron transport chain. 

By means of an ionophore specific for potassium (e.g. vahnomycin) or for protons (tetrachlorsalicylaniUde, TCS), Sachs et al. showed that the exchange process is electroneutral. The proton gradient generated by addition of ATP to the vesicles slowly dissipates due to the ion permeability of the vesicle. Addition of TCS leads to some increase in the rate of proton efflux, but a much faster proton release is observed upon addition of vahnomycin (Fig. 4). This means that the permeability of the vesicle for K is lower than for [66,78]. The ionophore nigericin, which exchanges H + for K, completely abolishes the proton gradient [58]. 

Fig. 11. Synthesis of ATP by substrate level phosphorylation and the generation of A/ii] by lactate-proton efflux during honaolactic fermentation.

Indirect methods of measuring channel transport are based on measurements of indicators trapped within a vesicle bilayer membrane. " These measurement methods include Na-NMR UV/Vis or fluorescence spectroscopy of pH. metal ion concentration, or concentration of environment-sensitive indicators radiochemical techniques and pH stat techniques to monitor proton efflux. These methods give evidence that an active compound can alter membrane permeability, but it is difficult to establish a distinction between channel function, transport via carriers, or a nonspecific membrane disruption. ... 

CORRELATION BETWEEN BUFFERING CAPACITY AND PROTON EFFLUX IN BACTERIAL CHROMATOPHORES... 

V/S kjjj, where V and S are the volume and surface of the vesicles and k is the first order kinetic constant for the proton efflux ... 

We have shown that in chromatophores the membrane-bound FOFl-ATPase is inacive, and must be activated by an artificially applied -, or light-induced Apj + [6.3]. Thus one might argue that in proteoliposomes the threshold Apjj+ of 90 mV for the onset of ATP-synthesis is a reflection of this activation requirement. However we have shown that in proteoliposomes the FOFl-ATPase activity was activated to nearly the maximum extent by application of a -diffusion potential of 77 mV [2]. Thus at least in our system the activation requirement of the enzyme does not explain the threshold Apjj+ of 90 mV for the onset of ATP-synthesis. As suggested by Brune et al. [9], the reason for this may be that the rate of ATP-synthesis is determined by the rate of proton efflux across the membrane. This rate in turn was found to exhibit an exponential dependence on the transmembrane AaH+-... 

In addition, we have taken into account the proton efflux between the different flashes of the group. Numerical integration of this curve then leads to the amount of protons translocated across the membrane due to ATP synthesis,... 

Earlier measurements with the same method resulted in H "/ATP = 3. The measurements reported above differ in two respects (1) The proton efflux between the different... 

Correlation Between Buffering Capacity and Proton Efflux in Bacterial Chromatophores 85 M.P. Turina, G. Venturoli, B.A. Melandri... 

Gonzalez-Reyes et al (1992) have reported that APR induces a quick and permanent plasmalemma hyperpolarization and stimulates the proton efflux in onion roots. Ascorbate and DHA also induce hyperpolarization and acidification of media although their effects are transient. More recently, Gonzalez-Reyes et al (1994a) reported that ascorbate can also stimulate root elongation when culture conditions allow ascorbate to be oxidized in an optimal way to produce APR. On the contrary, inhibition of ascorbate oxidation leads to an inhibition of ascorbate-mediated acceleration of root elongation. 

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