Proton efflux measurements

The proton pumping activity of Candida species was determined by monitoring acidification of external medium by measuring pH as described previously [63,64]. Briefly, mid-log phase cells harvested from YPD medium were washed twice with distilled water and routinely 0.1 g cells were suspended in 5 ml solution containing 0.1 M KCl, 0.1 mM CaClj in distilled water. Suspension was kept in a double-jacketed glass container with constant stirring. The container was connected to a water circulator at 25 C. Initial pH was adjusted to 7.0 using 0.01 M HCl/NaOH. Test materials were added to 

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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). 

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. 

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). 

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. ... 

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... 

Besides the remarks made above, which might notably weaken one s confidence in the electrogenic proton pump theory, let us stress some further complications. First, it is impossible to perform direct proton permeability measurements by the use of tritium in radiotracer methods. Second, several processes might be correlated to the external pH change, like effects caused by CO2 transport, 0H efflux, HCO5 uptake, the permeability of H and ions via pores, and kinetic control of all ionic pumps. 

Proton efflux in the dark with Anabaena variabilis, induced by an oxygen pulse, is shown in Figure lA. The efflux decreased after the oxygen had been consumed by respiration. Subsequently, a slow uptake of protons was measured (dotted line of part A). No major differences were seen using other blue-green algae (e.g. Anacystis, Aphanocapsa, Nostoc). 

Compiling the K /e ratios of mitochondria and of several bacterial species from the literature, values of 2 to 6 are found. The P/e ratio of blue-green algae, however, is comparable to other organisms. Obviously, in blue-greens, only a minor part of the electrons moving along the respiratory chain mediate proton efflux out of the cell. We conclude from our data that most of the respiratory electron transport is located on the thylakoid membrane. Thus, proton translocation driven by thylakoid-located electron flow cannot be determined as external acidification by intact cells, which leads to the low H /e ratios as measured. 

ATPase also catalyzed a passive Rb -Rb exchange, the rate of which was comparable to the rate of active Rb efflux. This suggested that the K-transporting step of H,K-ATPase is not severely limited by a K -occluded enzyme form, as was observed for Na,K-ATPase. Skrabanja et al. [164] also described the reconstitution of choleate solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. With the use of a pH electrode to measure the rate of H transport they observed not only an active transport, which is dependent on intravesicular K, but also a passive H exchange. This passive transport process, which exhibited a maximal rate of 5% of the active transport process, could be inhibited by vanadate and the specific inhibitor omeprazole, giving evidence that it is a function of gastric H,K-ATPase. The same authors demonstrated, by separation of non-incorporated H,K-ATPase from reconstituted H,K-ATPase on a sucrose gradient, that H,K-ATPase transports two protons and two ions per hydrolyzed ATP [112]. 

Under these conditions, a typical measured proton flux might be in the range of 10 15 mol/(cm2 s). To compare this value with that of potassium, 1 M potassium ion (as potassium sulfate) could be trapped inside the same liposomes, and potassium efflux into 1 M choline sulfate could be measured with a potassium-sensitive electrode. A typical result might again be in the range of 10 15 mol/(cm2 s). The proton permeability anomaly now becomes clear The same flux is measured for both potassium ion and protons, yet the proton flux is driven by a concentration of protons 6 orders of magnitude less than the concentration of potassium ions. Estimates of the relative permeabilities of the bilayer to protons and potassium using these flux data yield values of 10 6 cm/s for protons and 10 12 cm/s for potassium ion. 

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