The fluorescence intensity of the lipophilic probe N-phenyl-1-naphthylamine responds to the oxidation-reduction state of purified Escherichia coli cytochrome o incorporated into phospholipid vesicles

N-Phenyl-1-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to the redox state of purified cytochrome o incorporated into lipid vesicles formed from purified or E. coli phospholipids. NPN was bound to the proteoliposomes to produce a steady-state level of fluorescence intensity. Addition of the substrate ascorbate, in the presence of phenazine methosulfate as an electron donor, did not alter the fluorescence. However, following complete removal of oxygen from the medium by oxidation of the substrate by molecular oxygen catalyzed by cytochrome o, there was an increase in the fluorescence of NPN. This coincided with the reduction of cytochrome o. Reoxidation of the cytochrome by addition of oxygen decreased the fluorescence to steady-state levels until the oxidant had been completely reduced. The fluorescence changes were dependent on the incorporation of cytochrome o into phospholipid vesicles but were insensitive to the state of energization of the vesicle membrane.     

Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) as a probe of internal aqueous hydrogen ion concentration in phospholipid vesicles

The fluorescence intensity (at 510 nm) of the hydrophilic pyrene analogue 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) is strongly dependent upon the degree of ionization of the 8-hydroxyl group (pKa = 7.2) and hence upon the medium pH, over the range pH 6--10. Because of its polyanionic character, pyranine does not bind significantly to phospholipid vesicles having a net anionic surface charge. As a result, it is possible to form vesicles in the presence of pyranine which, after removal of external probe by gel filtration, contain pyranine entrapped within the internal aqueous compartment. Once entrapped, pyranine does not readily leak out of the vesicles. Because the fluorescence properties of entrapped pyranine resemble closely the properties of bulk pyranine solution with respect to pH sensitivity, pyranine can be used as a reliable reporter of aqueous pH changes within anionic vesicles. When HCl is rapidly added to a suspension of unilamellar soybean phospholipid (asolectin) vesicles preincubated at alkaline pH, a biphasic decrease in the pH of the vesicle inner aqueous compartment is observed. An initial, very rapid and electrically uncompensated H+ influx (t 1/2 less than 1 s) results in the generation of a transmembrane electric potential opposing further H+ influx. This leads to the development of a much slower (t 1/2 approximately equal to 5 min), valinomycin-sensitive, proton--counterion exchange which continues until the proton concentration gradient is eliminated. Similar results were obtained in asolectin vesicles prepared by detergent dilution, in sonicated egg phosphatidylcholine vesicles, and in multilamellar asolectin liposomes. The rather high permeability of soybean lipid membranes to H+ is surprising in view of the widespread use of these lipids for the reconstitution of membrane proteins which are thought to generate or utilize H+ ion gradients in energy transduction reactions.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic probe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, D-lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Transmembrane pH gradients and functional heterogeneity in reconstituted vesicle systems

The pH-sensitive, membrane impermeant fluorescence probes 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine; pK_a = 7.2) and 1-naphthol-3,6-disulfonate (Naps pK_a = 8.2) can be simultaneously entrapped within the intravesicular aqueous compartment of unilamellar vesicles and reconstituted proteoliposomes, where they function as reliable reporters of the intravesicular pH. Because the two probes are sensitive to pH over different but overlapping ranges, the useful monitoring range for the co-trapped probe pair extends from pH 6.5 to 9. In vesicles pre-equilibrated at a given pH and then subjected to a sudden change in external pH, the rate and extent of the subsequent change in internal pH are identical at all times during the re-equilibration, regardless of which probe is used to monitor the change. However, in reconstituted bacteriorhodopsin proteoliposomes, the size of the transmembrane pH gradient generated in the light always appears greater when pyranine is used to monitor internal pH. This discrepancy can most readily be understood in terms of heterogeneity in the vesicle suspension, with at least two populations of vesicles, one active in proton and one inactive. A simple algorithm was developed which generates, from the observed internal pH changes for two probes of different pK_a, the percentage of vesicles which are inactive, as well as the actual internal pH of the active fraction. The applicability of this algorithm was subsequently confirmed using a suspension of vesicles in which the level of heterogeneity was deliberately altered by the addition of various amounts of gramicidin. The apparent transmembrane pH gradient for the vesicle population as a whole decreased with increasing gramicidin, as did the calculated percentage of vesicles able to maintain a pH gradient, while the transmembrane gradient calculated for the active vesicle fraction only was essentially unaffected by gramicidin.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.     

Fatty acid flip-flop in a model membrane is faster than desorption into the aqueous phase

Fatty acids (FA) are known to diffuse (flip-flop) rapidly across protein-free phospholipid bilayers in their un-ionized form. However, whether flip-flop through the hydrophobic core of the bilayer or desorption from the membrane into the aqueous phase is the rate-limiting step in FA transport through membranes is still debated. The issue has remained unresolved in part by disagreements over whether some methods of adding FA create artifacts that lead to erroneous conclusions and in part by the lack of fluorescence methods to monitor each individual step. Here we study the kinetics of FA transfer from donors to phospholipid vesicles (small and large unilamellar vesicles) by a dual fluorescence approach that utilizes the probes fluorescein phosphatidylethanolamine (FPE) and pyranine. FPE detects the concentration of FA anions in the outer membrane leaflet, allowing a precise measurement of kinetics of FA adsorption or desorption. Our results showed that as soon as FPE detects adsorption of FA into the outer leaflet, pyranine detects its movement to the inner leaflet. We further demonstrated that (i) flip-flop for FA with 14-22 carbons is much faster than the rates of desorption and therefore cannot be the rate-limiting step of FA translocation across membranes; (ii) fluorescence changes detected by probes located on or in acceptor vesicles are dependent upon the method used to deliver the FA (i.e., uncomplexed, or complexed to albumin or phospholipid bilayers); however, (iii) transfer kinetics observed in the presence of different donors is rate-limited by the desorption of FA from the donor into the aqueous phase rather than by flip-flop.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b₅ has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b₅vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b₅ upon the interaction with its redox partners is discussed.     

Temperature dependence of 1,6-diphenyl-1,3,5-hexatriene fluorescence in phophoslipid artificial membranes.

The fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles is a function of the physical state of the lipid. Below the phase transition, the polarization approaches the theoretical maximum for total immobilization while above the phase transition the fluorescence becomes nearly completely depolarized. The discontinuity in the temperature dependence of polarization occurs within a temperature range under 5 degrees C in the case of pure phospholipids, but for mixed phospholipids occurs over a temperature range greater than 20 degrees C. From these data, phase diagrams describing the gel-sol equilibrium can be constructed; the phase diagrams correspond well with those described in the literature which were constructed using spin-label probes or from x-ray diffraction patterns. The marked change in polarization at the phase transition may be related to the packing of the probe molecule into the lipid bilayer: fluorescence measurements on oriented bilayers indicate that below the phase transition the long axis of the probe is oriented perpendicular to the plane of the membrane while above the transition the probe is oriented randomly relative to the plane of the membrane.     

Redox-linked proton translocation in the b-c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. General characteristics and control of electron flow by delta micro H+

A study is presented of the characteristics of redox-linked proton translocation in the b-c1 complex isolated from beef-heart mitochondria and reconstituted into phospholipid vesicles. Measurements of the H+/e- stoichiometry, with three different methods, show that four protons are released from the vesicles per 2e- flowing from quinols to cytochrome c, two of these protons formally deriving from scalar oxidation of quinols by cytochrome c. This H+/e- stoicheiometry is independent of the initial redox state of the b-c1 complex (fully reduced or oxidized) and the rate of electron flow through the complex. It does not change in the pH range 6.0 - 7.2, but declines to 1.5 going with pH from 7.2 - 8.3. This decrease is accompanied by enhancement of the rate of electron flow in the coupled state. Collapse of delta psi effected by valinomycin addition to turning-over b-c1 vesicles resulted in substantial oxidation of cytochrome b-566 and comparable reduction of cytochrome c1, with little oxidation of cytochrome b-562. Nigericin alone had no effect on the steady-state redox levels of b and c cytochromes. Its addition in the presence of valinomycin caused oxidation of b cytochromes but no change in the redox state of cytochrome c1. Valinomycin alone caused a marked enhancement of the rate of electron flow through the complex. Nigericin alone was ineffective, but caused further stimulation of electron flow when added in the presence of valinomycin. The data presented are discussed in terms of two mechanisms: the Q cycle and a model based on combination of protonmotive catalysis by special bound quinone and proton conduction along pathways in the apoproteins.     

Change of synaptic membrane lipid composition and fluidity by chronic administration of lithium

The effect of chronic administration of lithium salts on the lipid composition and physical properties of the synaptosomal plasma membrane was examined in rat brain. The effect of lithium treatment has been studied on the fluorescence polarization of synaptosomal plasma membrane and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization of lipophilic probes was used to study membrane lipid structure. Steady-state polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe of the hydrophobic core, was significantly lower in plasma membranes from lithium-treated animals. Altered DPH polarization was due to a decrease in the order parameter of the probe. The lithium-treatment also changed the fluorescence of 1-anilino-8-naphthalene sulfonate (ANS), a probe that binds to the polar head group of the phospholipids and to proteins on the membrane surface. Synaptic plasma membranes from treated rats presented no significant changes on the cholesterol-to-phospholipid ratio, although the phospholipid class distribution was altered and the membrane phospholipid unsaturation increased. In summary, the neural plasma membranes became disorder after chronic lithium administration at therapeutic levels. This structural change may be due to changes in plasma membrane phospholipid distribution and to the degree of unsaturation of phospholipid fatty acids.     

Molecular mechanism of general anesthesia: I. Fluorescence studies in mitochondrial membranes

We have tested the working hypothesis that anesthetics, by labilizing lipid-protein interactions, induce conformational changes in membrane proteins involved in the transmission of neural impulses. In the first communication of this series we report that general anesthetics induce changes in the fluorescence of the probes ANS and NPN in model membranes, lipid vesicles and mitochondria. The changes observed concern the quantumyield but not the position of the emission maximum. Such changes may be interpreted as due to fluidization of the membrane core (NPN), accompanied by variable effects in the membrane surface(ANS).     

Characterization of the fluorophore 4-heptadecyl-7-hydroxycoumarin: a probe for the head-group region of lipid bilayers and biological membranes

The fluorophore 4-heptadecyl-7-hydroxycoumarin was used as a probe to study the properties of phospholipid bilayers at the lipid-water interface. To this end, the steady-state fluorescence anisotropy, the differential polarized phase fluorometry, and the emission lifetime of the fluorophore were measured in isotropic viscous medium, in lipid vesicles, and in the membrane of vesicular stomatitis virus. In the isotropic medium (glycerol), the probe showed an increase in the steady-state fluorescence anisotropy with a decrease in temperature, but the emission lifetime was unaffected by the change in temperature. In glycerol, the observed and predicted values for maximum differential tangents of the probe were identical, indicating that in isotropic medium 4-heptadecyl-7-hydroxycoumarin is a free rotator. Nuclear magnetic resonance and differential scanning calorimetric studies with lipid vesicles containing 1-2 mol % of the fluorophore indicated that the packaging density of the choline head groups was affected in the presence of the probe with almost no effect on the fatty acyl chains. The fluorophore partitioned equally well in the gel and liquid-crystalline phase of the lipids in the membrane, and the phase transition of the bilayer lipids was reflected in the steady-state fluorescence anisotropy of the probe. The presence of cholesterol in the lipid vesicles had a relatively small effect on the dynamics of lipids in the liquid-crystalline state, but a significant disordering effect was noted in the gel state. One of the most favorable properties of the probe is that its emission lifetime was unaffected by the physical state of the lipids or by the temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the location of fluorescent probes attached to fatty acids using parallax analysis of fluorescence quenching: effect of carboxyl ionization state and environment on depth.

In this report, parallax analysis of fluorescence quenching (see the preceding paper in this issue) was used to determine the location (depth) of anthroyloxy and carbazole probes attached to model membrane inserted fatty acids. A monotonic increase in depth was found as the number of carbon atoms between the attachment site of the probe and the fatty acyl carboxyl group is increased. It was also found that depth is sensitive to pH, with an increase in probe depth upon protonation of the fatty acid carboxyl group of around 0.5-2.5 A, depending on probe location and identity. This result shows that carboxyl protonation causes an increase in depth all along a fatty acid chain. In addition, it indicates that parallax analysis is very sensitive to small changes in depth. At a given pH, no significant change in probe depth was observed in vesicles containing anionic phospholipid or at various ionic strengths, suggesting these parameters do not strongly regulate fatty acyl chain location. It was also found that there is a decrease of the apparent depth of each of the fatty acyl attached probes both at longer excitation wavelengths and at longer emission wavelengths. This is consistent with there being a distribution of depth for each fluorophore, with shallower fluorophore dominating the fluorescence at red-shifted wavelengths. Solvent relaxation effects also appear to contribute to this wavelength dependence.     

Assay of Proton-Coupled Glycolate and D-Glycerate Transport into Chloroplast Inner Envelope Membrane Vesicles by Stopped-Flow Fluorescence

The transport of glycolate and D-glycerate across the inner envelope membrane of intact chloroplasts is rapid and mediated by a translocator with proton/substrate symport activity. The true initial rate of glycolate or D-glycerate transport could not be measured by conventional methods. To resolve the initial rates of glycolate and D-glycerate transport, a stopped-flow fluorescence assay was developed that allows the indirect observation of transport from about 4 ms after mixing. Inner envelope vesicles from pea (Pisum sativum) or spinach (Spinacia oleracea) chloroplasts were loaded with the fluorescent pH indicator pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) by a freeze-thaw sonication protocol. A rapid quenching of pyranine fluorescence was detected after mixing the vesicles with either glycolate or D-glycerate. This quenching was the result of acidification of the interior of the vesicles. D-Glycerate- or glycolate-induced acidification displayed saturation kinetics and was inhibited by pretreatment of the vesicles with N-ethylmaleimide. D-Glycerate was more effective than L-glycerate in causing the pH decrease. Also, L-mandelate inhibited D-glycerate-induced acidification much more strongly than D-mandelate. The glycolate/D-glycerate-induced pH decrease is consistent with glycolate/D-glycerate translocator activity. The assay was placed on a quantitative basis by converting fluorescence changes to pH and measuring the internal buffering capacity of the vesicles. The rates of transport across the inner envelope membrane were estimated to be as fast, if not faster, than those of transport in intact chloroplasts.     

pH determination by pyranine: medium-related artifacts and their correction

Pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) is a water-soluble, membrane-impermeable fluorophore having fluorescent excitation and emission spectra that are highly dependent on medium pH. This combination makes it one of the most commonly used pH-sensitive fluorescent probes to monitor pH and pH changes in biochemical and biophysical research. The pK(a) of this probe is reported to be approximately 7.3, but several studies (including the current one) have shown that this value varies with medium composition. If this is not taken into account, pH determinations based on pyranine may be misleading. We found that in the presence of salts, pK(a) is shifted downward to lower values; therefore, the calculated pH is shifted upward relative to the actual pH as determined by a pH meter. This shift is a consequence of both the type and the concentration of anions and cations that form the salt. Divalent cations cause a larger upward shift in the calculated pH than do monovalent cations. Of all the salts tested, ammonium sulfate has the least effect, and calcium perchlorate has the largest effect, on the pH value calculated by pyranine. Salts are not the only species that affect the pK(a) of pyranine. The presence of the polymer polyethylene glycol (PEG) induces an effect opposite to that of salt (i.e., an upward pK(a) shift), which is expressed as pH being more acidic than that measured by a pH meter. Another nonelectrolyte, dextrose, has no such effect. The effect of both cations and anions can be explained based on their order in the Hofmeister series, whereas the effect of PEG is explained by its high water-binding capacity. Both the ions and PEG change the structure of water and its interaction with pyranine, thereby changing pyranine's apparent pK(a).     

Effect of membrane potential and pH gradient on electron transfer in cytochrome oxidase

Steady-state spectra of cytochrome oxidase in phospholipid vesicles were obtained by using hexaammineruthenium(II) and ascorbate as reductants. Cytochrome a was up to 80% reduced in the steady state in coupled vesicles. Upon addition of nigericin or acetate, which decrease delta pH, resulting in an increase in delta psi, cytochrome a became more oxidized in the steady state with no change in the rate of respiration. On the other hand, uncouplers or valinomycin plus nigericin, which lower both delta psi and delta pH, stimulated respiration 2-8-fold and also lowered the steady-state level of reduction of cytochrome a. These experiments indicate that electron transfer between cytochromes a and a 3 is sensitive primarily to the pH gradient. Studies with the reconstituted and the soluble enzyme at various pH values indicated that the pH on the matrix side of the membrane, rather than delta pH, controlled the steady-state level of reduced cytochrome a. Hexaammineruthenium(II) substituted for cytochrome c in measurements of proton pumping by cytochrome oxidase. Dicyclohexylcarbodiimide, which eliminated proton pumping by cytochrome oxidase, decreased the effect of ionophores on the steady-state level of reduced cytochrome a.     

Fusion of Spiroplasma floricola cells with small unilamellar vesicles is dependent on the age of the culture.

Small unilamellar vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride and mixed with intact Spiroplasma floricola cells. The increase in fluorescence observed was interpreted as a result of the dilution of the probe in the unlabeled S. floricola membranes because of lipid mixing upon fusion. The progression of S. floricola cultures to the stationary phase of growth was accompanied by a sharp decrease in the ability of the cells to fuse with small unilamellar vesicles. Low fusogenic activity was also detected in cells from cultures that were aged in a growth medium maintained at pH 7.5 throughout the growth cycle. Chemical analysis of the cell membrane preparations isolated from cells harvested at the various phases of growth revealed that the phospholipid content and composition and the cholesterol/phospholipid molar ratio were changed very little upon aging of the cultures. Likewise, no changes in the fatty acid composition of membrane lipids were detected, with palmitic and oleic acids predominating throughout the cycle. Nonetheless, upon aging of S. floricola cultures, a pronounced increase in the levels of both cholesteryl esters, incorporated from the growth medium, and organic peroxides was observed. A decrease in both fluorescence anisotropy of diphenylhexatriene and merocyanine 540 binding to membranes of aged cells was also detected. The possible influence of these changes on the fusogenic activity of the cells is discussed.     

pH-induced reorganization of synaptic membrane as revealed by fluorescence anisotropy and energy transfer

Two fluorescent probes, 2-(9-anthroyloxy)stearate and 12-(9-anthroyloxy)stearate, were used to investigate the effects of the neutralization of membrane charges on the organization of synaptic plasma membrane. Steady state fluorescence anisotropy measurements showed that a pH decrease provoked a rigidification of the synaptic membrane surface, whereas the bilayer core remained unaffected. The same effect was observed with negatively charged lipid vesicles. The relative distribution of proteins and the probes was estimated by fluorescence energy transfer from protein tryptophans to fluorescent probes: a pH decrease provoked an increase of the energy transfer, which was most pronounced with the surface probe, indicating an average closer packing between proteins and the probes. The modifications induced by a pH decrease were temperature dependent and were most marked at low temperatures. The results suggest that neutralization of the membrane charges provoked a redistribution of both membrane lipids and proteins. These findings are discussed in terms of a heterogeneous distribution of these membrane components.     

A study of H+ transport in gastric microsomal vesicles using fluorescent probes.

Fluorescent amines, 9-aminoacridine, acridine orange and quinacrine, were used as probes for a pH gradient (deltapH) across gastric microsomal vesicles. Analysis of probe uptake data indicates that 9-aminoacridine distributes across the membrane as a weak base in accordance with the deltapH. On the other hand, acridine orange and quinacrine show characteristics of binding to membrane sites in addition to the accumulation in response to deltapH. A discussion of the advantages and limitations of the probes is presented. Application of these probes to pig gastric microsomal vesicles indicates that that K+-stimulated ATPase is responsible for the transport of H+ into the vesicles and thus develops a deltapH across the membrane. The deltapH generated by the K+-ATPase has a definite requirement for internal K+. The proton gradient can be discharged slowly after ATP depletion or rapidly either by detergent disruption of the vesicles or by increasing their leakiness using both H+ and K+ ionophores. On the other hand, the sole use of the K+ ionophore, valinomycin, stimulates the ATP-induced formation of deltapH by increasing the availability of K+ to internal sites. This stimulation by valinomycin requires the presence of permeable anions like Cl-. Analysis of the Cl- requirement indicates that in the presence of valinomycin the net effect is the accumulation of HCl inside the gastric vesicles. With an external pH of 7.0, the ATP-generated deltapH was calculated to be from 4 to 4.5 pH units. The results are consistent with the hypothesis that the K+-stimulated ATPase drives a K+/H+ exchange across the gastric vesicles. Since other lines of evidence suggest that these gastric microsomes are derived from the tubulovesicular system of the oxyntic cell, the participation of the ATP-driven transport processes in gastric HCl secretion is of interest.