Net Proton Flux Research Articles

37] Electron paramagnetic resonance methods for measuring H+/OH− fluxes across phospholipid vesicles

Publisher Summary This chapter describes the application of magnetic resonance probe techniques to measure net proton flux and permeability and to obtain current–voltage characteristics for H + /OH – diffusion across small model membrane vesicle systems. Lipid vesicles are easily manipulated and reproducible and provide an ideal model system to make these measurements. The EPR probe methodology allows accessing the values of both electrical and chemical gradients across these vesicles. These methods are sensitive and allow the measurement of current densities on the order of a few picoamps/cm 2 or less. Estimating the H + /OH – permeability of small lipid vesicles are accomplished in several ways and many different methods are employed. The two procedures described in the chapter use different approaches. In the first procedure, transmembrane voltages (∆ ψ ) that develop following the establishment of a pH gradient are measured. In the second procedure, the H + /OH – flow is estimated by measuring the change in ∆ ψ , following the establishment of ∆pH under weakly buffering conditions. These two procedures provide an unambiguous measurement of electrogenic and neutral H + /OH – flow.

Kinetics and pH-dependence of glycine-proton symport in Saccharomyces cerevisiae

Interactions between intercellular pH (pH i) and H + -coupled transmembrane transport of glycine have been studied by means of 31P-NMR, using both aerobic and ‘energy starved’ cells of the yeast Saccharomyces cerevisiae. The general features of glycine transport in the yeast strain used (NCYC 239) are similar to those already reported for Saccharomyces carlsbergensis and S. cerevisiae, there being two kinetically distinct glycine uptake systems, with pH-independent K 1 2 values near 14 and 0.4 mM, respectively, but pH-dependent maximal velocities. Glycine transport itself has no measurable effect on pH i in aerobic cells, and only a marginal effect in energy-starved cells, but changes of pH i, imposed by extracellular addition of butyric acid, strongly influence glycine transport. Indeed, the dependence of glycine influx (in energy-starved cells) upon cytoplasmic H + concentration appears to be third order, showing Hill slopes of 2.7–3.0. A crucial kinetic role for cytoplasmic pH in glycine transport is further indicated by a proportionality between the decline of flux and the decline of pH i produced by various metabolic inhibitors and uncouplers. Extracellular pH (pH o, by contrast, has only a weak effect on glycine influx, showing a Hill slope of 0.5. The major observations can be accommodated by a simple cyclic carrier scheme, in which 2 or more protons are transported along with glycine, but only one extracellular proton binding site dissociates in the testing range, with a p K near 5.5. The model requires a finite membrane potential, which must be somewhat sensitive to both pH i and pH o, and accomodates the discrepancy between measured net proton flux (one per glycine) and the kinetically required proton flux (two or more per glycine) by shunting through other proton-conducting pathways in the yeast membrane.

Perfusion of onion root xylem vessels: a method and some evidence of control of the pH of the xylem sap

We describe a method for perfusing the xylem in the stele of excised onion roots with solutions of known composition under a pressure gradient. Tracer studies using [(14)C] polyethylene glycol 4000 and the fluorescent dye, Tinopal CBSX, indicated that perfusing solutions passed exclusively through the xylem vessels. The conductance of the xylem was small over the apical 100 mm of the root axis but increased markedly between 100 and 200 mm. Unbuffered perfusion solutions supplied in the range pH 3.7-7.8 emerged after passage through the xylem adjusted to pH 5.2-6.0, indicating the presence of mechanisms for absorbing or releasing protons. This adjustment continued over many hours with net proton fluxes apparently determined by the disparity between the pH of the perfusion solution and the usual xylem sap pH of about 5.5. Mild acidification of the xylem sap by buffered perfusion solutions increased the release of (86)Rb (K(+)) and (35)SO4 (2-) from the stelar tissue into the xylem stream. The ion-transporting properties of onion roots seemed little changed by excision from the bulbs, or by removal of the apical zones of the root axis. The pH of sap produced by root pressure resembles that found in the outflow solutions of perfused root segments.

Change in Proton Extrusion in Excised Root Tips of Zea mays Seedlings during “Ageing”

Summary If excised 3 mm long root tips of Zea mays were shaken in 2 mM CaCl 2 , their ability to acidify the medium decreased quickly. This phenomenon was nullified by adding D-glucose to the preincubation medium and did not depend on the ion composition of the buffer in which the proton fluxes were measured. The net proton fluxes are largely determined by the consumption or formation of acid in metabolism. A causal connection with other “ageing” phenomena (increased absorption capacity) could not be established.

Control of intracellular pH. Predominant role of oxidative metabolism, not proton transport, in the eukaryotic microorganism Neurospora.

Recessed-tip microelectrodes were used to measure internal pH (pHi) in the fungus Neurospora, and to examine the response of pHi to several kinds of stress: changes of extracellular pH (pHo), inhibition of the principal proton pump in the plasma membrane, and inhibition of respiration. Under control conditions, at pHo = 5.8, pHi in Neurospora is 7.19 +/- 0.04. Changes of pHo between 3.9 and 9.3 affect pHi linearly but with a slope of only approximately 0.1 unit pHi per unit pHo, stable pHi being reached within 3 min of changed pHo. Despite a postulated high passive permeability of the Neurospora membrane to protons (Slayman, 1970), neither active nor passive H+ transport appears critical to pHi because (alpha) specific inhibition of the proton pump by orthovanadate has little effect on pHi, and (b) cytoplasmic acidification produced by respiratory blockade is unaffected by the size or direction of proton gradient. To convert measured changes in pHi into net proton fluxes, intracellular buffering capacity (beta i) was measured by the weak acid/weak base technique. At pHi = 7.2, beta i was (-) 35 mmol H+ (liter cell water)-1 (pH unit)-1, but beta i increased substantially in both the acid and alkaline directions, which suggests that amino acid side chains are the principal source of buffer.

Coupled transport of protons and anions through lipid bilayer membranes containing a long-chain secondary amine.

Transport of protons and halide ions through planar lipid bilayers made from egg lecithin and a long-chain secondary amine (n-lauryl [trialkylmethyl] amine) in n-decane was studied. Net proton fluxes were measured with a pH electrode, and halide fluxes were measured with 82Br- and 36Cl-. In membranes containing the secondary amine, a large net proton flux was produced either by a Br- gradient with symmetrical pH or by a pH gradient with symmetrical Br-, but not by a pH gradient in Br--free solutions. This H+ flux was electrically silent (nonconductive), and the H+ permeability coefficient was greater than 10(-3) cm sec-1 in 0.1 M NaBr. In Br--free solutions, H+ selectivity was observed electrically by measuring conductances and zero-current potentials generated by H+ activity gradients. The permeability coefficient for this ionic (conductive) H+ flux was about 10(-5) cm sec-1, several orders of magnitude smaller than the H+ permeability of the electroneutral pathway. Large electroneutral Br- exchange fluxes occurred under symmetrical conditions, and the permeability coefficient for Br- exchange was about 10(-3) cm sec-1 at pH 5. The one-way Br- flux was inhibited by substituting SO4= for Br- on the "trans" side of the membrane. These results support a "titratable carrier" model in which the secondary amine exists in three forms (C, CH+ and CHBr). Protons can cross the membrane either as CHBr (nonconductive) or as CH+ (conductive), whereas Br- crosses the membrane primarily as CHBr (nonconductive). In addition to these three types of transport, there is also a pH-dependent conductive flux of Br- which has a permeability coefficient of about 10(-7) cm sec-1 at pH 5. Experiments with lipid monolayers suggest that the pH dependence of this conductive flux is caused by a change in surface potential of about +100 mV between pH 9.5 and 5.0.

Phosphorus-31 nuclear magnetic resonance studies of active proton translocation in chromaffin granules.

ATP hydrolysis and proton translocation in chromaffin granules were followed using 31P nuclear magnetic resonance. The intragranular pH affects the resonance frequency of the gamma-phosphate of granular ATP. By measuring frequency vs. pH in solutions which simulate the intragranular matrix, this may be calibrated to give quantitative pH measurements. The pH in the resting granule is 5.65 +/- 0.15. This drops by 0.4 to 0.5 pH unit when ATP is added externally and protons are actively pumped into the granules. Because of differences in the composition and pH of the internal and external solutions, the resonances of internal and external nucleotides and Pi can be distinguished. Consequently, ATP hydrolysis and changes in internal pH may be observed simultaneously and continuously in a single sample of chromaffin granules. From the measured buffering capacity of a reconstituted intragranular solution, pH changes were converted into an absolute number of protons translocated. The net proton flux (protons translocated/ATP hydrolyzed) was about 1.0 immediately after external ATP addition but fell toward zero as the pH gradient increased to a new steady state. These 31P NMR results agree with intragranular pH measurements determined from methylamine distribution and with H+/ATP stoichiometries calculated from pH changes observed in the external medium.

Cell Potentials, Cell Resistance, and Proton Fluxes in Corn Root Tissue

Studies were made of the effect of dithioerythritol on net proton flux, potassium influx and efflux, cell potential, and cell resistance in fresh and washed corn (Zea mays L. WF9XM14) root tissue. Dithioerythritol induces equal proton influx and potassium efflux rates, decreases membrane resistance, and hyperpolarizes the cell potential. Greater effects on H(+) and K(+) fluxes are secured at pH 7 than at pH 5. Other sulfhydryl-protecting reagents produced the same responses. No evidence could be found that dithioerythritol affected energy metabolism or membrane ATPase, and proton influx was induced in the presence of uncoupling agents.We deduce that dithioerythritol activates a passive H(+)/K(+) antiport, driven in these experiments by the outwardly directed electrochemical gradient of K(+). The net effect on H(+) and K(+) fluxes is believed to reside with the combined activity of a polarized H(+)/K(+) exchanging ATPase and the passive H(+)/K(+) antiport. A model is presented to show how the combined system might produce stable potential differences and K(+) content.