Ion gradient-induced membrane translocation of model peptides.

The K+ diffusion potential-induced association of synthetic model peptides carrying a single positive charge originating from the NH2-terminal amino function with large unilamellar vesicles (LUV) consisting of phosphatidylcholine (PC) has been reported previously (de Kroon, A. I. P. M., J. de Gier, and B. de Kruijff. 1989. Biochim. Biophys. Acta. 981:371-373). To determine the vesicle localization of the associated peptides, fluorescence measurements utilizing the peptides' tryptophan residue as intrinsic fluorescent probe were performed. The application in these measurements, of vesicles that exhibit an asymmetric transbilayer distribution of brominated PC which is a quencher of tryptophan fluorescence, unequivocally demonstrated that the peptide H3N(+)-AIMLWA-Ome (AIXme+) is accumulated in the interface of the inner leaflet of the vesicle membrane in response to the valinomycin-induced K+ diffusion potential (negative inside). The relative contributions of the membrane potential (delta psi) and the pH gradient (delta pH, acidic inside) induced by the K+ diffusion potential, to the process have been assessed. An analysis of the pH and delta pH dependencies of the process demonstrated that the K+ diffusion potential-induced peptide accumulation is largely determined by a redistribution of peptide according to the transbilayer pH gradient, in agreement with a translocation across the vesicle membrane of the neutral, deprotonated form of the peptide. The general validity of the mechanism proposed for the vesicle-uptake of AIXme+ has been examined by extending the experiments to peptide analogues with a single negative charge and to peptides with two positive charges, and by investigating the effect of incorporating the acidic phospholipid cardiolipin (CL) into the LUV. The incorporation of CL appeared not to affect the K+ diffusion, potential-induced vesicle uptake of AIXme+. The peptide H3N(+)-RMLWA-Ome (RXme2+) showed a small delta pH independent fluorescence response to the delta psi upon raising the CL content of the vesicles to 25%.     

Relationships between the Na+-H+ antiport activity and the components of the electrochemical proton gradient in Escherichia coli membrane vesicles

The kinetics of Na+ efflux from Escherichia coli RA 11 membrane vesicles taking place along a favorable Na+ concentration gradient are strongly dependent on the generation of an electrochemical proton gradient. An energy-dependent acceleration of the Na+ efflux rate is observed at all external pHs between 5.5 and 7.5 and is prevented by uncoupling agents. The contributions of the electrical potential (delta psi) and chemical potential (delta pH) of H+ to the mechanism of Na+ efflux acceleration have been studied by determining the effects of (a) selective dissipation of delta psi and delta pH in respiring membrane vesicles with valinomycin or nigericin and (b) imposition of outwardly directed K+ diffusion gradients (imposed delta psi, interior negative) or acetate diffusion gradients (imposed delta pH, interior alkaline). The data indicate that, at pH 6.6 and 7.5, delta pH and delta psi individually and concurrently accelerate the downhill Na+ efflux rate. At pH 5.5, the Na+ efflux rate is enhanced by delta pH only when the imposed delta pH exceeds a threshold delta pH value; moreover, an imposed delta psi which per se does not enhance the Na+ efflux rate does contribute to the acceleration of Na+ efflux when imposed simultaneously with a delta pH higher than the threshold delta pH value. The results strongly suggest that the Na+-H+ antiport mechanism catalyzes the downhill Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of ion gradients on the transbilayer distribution of dibucaine in large unilamellar vesicles

The uptake of dibucaine into large unilamellar vesicles in response to proton gradients (delta pH; inside acidic) or membrane potentials (delta psi; inside negative) has been investigated. Dibucaine uptake in response to delta pH proceeds rapidly in a manner consistent with permeation of the neutral (deprotonated) form of the drug, reaching a Henderson-Hasselbach equilibrium where [dibucaine]in/[dibucaine]out = [H+]in/[H+]out and where the absolute amount of drug accumulated is sensitive to the buffering capacity of the interior environment. Under appropriate conditions, high absolute interior concentrations of the drug can be achieved (approximately 120 mM) in combination with high trapping efficiencies (in excess of 90%). Dibucaine uptake in response to delta psi proceeds more than an order of magnitude more slowly and cannot be directly attributed to uptake in response to the delta pH induced by delta psi. This induced delta pH is too small (less than or equal to 1.5 pH units) to account for the transmembrane dibucaine concentration gradients achieved and does not come to electrochemical equilibrium with delta psi. Results supporting the possibility that the charged (protonated) form of dibucaine can be accumulated in response to delta psi were obtained by employing a permanently positively charged dibucaine analogue (N-methyldibucaine). Further, the results suggest that delta psi-dependent uptake may depend on formation of a precipitate of the drug in the vesicle interior. The uptake of dibucaine into vesicles in response to ion gradients is of direct utility in drug delivery and controlled release applications and is related to processes of drug sequestration by cells and organelles in vivo.     

Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients

Studies from this laboratory (Mayer et al. (1986) Biochim. Biophys. Acta 857, 123-126) have shown that doxorubicin can be accumulated into liposomal systems in response to transmembrane pH gradients (inside acidic). Here, detailed characterizations of the drug uptake and retention properties of these systems are performed. It is shown that for egg phosphatidylcholine (EPC) vesicles (mean diameter of 170 nm) exhibiting transmembrane pH gradients (inside acidic) doxorubicin can be sequestered into the interior aqueous compartment to achieve drug trapping efficiencies in excess of 98% and drug-to-lipid ratios of 0.36:1 (mol/mol). Drug-to-lipid ratios as high as 1.7:1 (mol/mol) can be obtained under appropriate conditions. Lower drug-to-lipid ratios are required to achieve trapping efficiencies in excess of 98% for smaller (less than or equal to 100 nm) systems. Doxorubicin trapping efficiencies and uptake capacities are related ito maintenance of the transmembrane pH gradient during encapsulation as well as the interaction between doxorubicin and entrapped citrate. This citrate-doxorubicin interaction increases drug uptake levels above those predicted by the Henderson-Hasselbach relationship. Increased drug-to-lipid ratios and trapping efficiencies are observed for higher interior buffering capacities. Retention of a large transmembrane pH gradient (greater than 2 units) after entrapment reduces the rate of drug leakage from the liposomes. For example, EPC/cholesterol (55:45, mol/mol) liposomal doxorubicin systems can be achieved which released less than 5% of encapsulated doxorubicin (drug-to-lipid molar ratio = 0.33:1) over 24 h at 37 degrees C. This pH gradient-dependent encapsulation technique is extremely versatile, and well characterized liposomal doxorubicin preparations can be generated to exhibit a wide range of properties such as vesicle size, lipid composition, drug-to-lipid ratio and drug release kinetics. This entrapment procedure therefore appears well suited for use in therapeutic applications. Finally, a rapid colorimetric test for determining the amount of unencapsulated doxorubicin in liposomal systems is described.     

Transmembrane distribution of lipophilic cations in response to an electrochemical potential in reconstituted cytochromec oxidase vesicles and in vesicles exhibiting a potassium ion diffusion potential

It has been shown previously that biogenic amines and a number of pharmaceutical agents can redistribute across vesicle membranes in response to imposed potassium ion or proton gradients. Surprisingly, drug accumulation is observed for vesicles exhibiting either a pH gradient (interior acidic) or a membrane potential (interior negative), implying that these compounds can traverse the lipid bilayer as either the neutral or charged species. This interpretation, however, is complicated by the fact that vesicles exhibiting a membrane potential (interior negative) accumulate protons in response to this potential, thereby creating a pH gradient (interior acidic). This raises the possibility that in both vesicle systems drug redistribution occurs in response to the proton gradient present. We have therefore compared the uptake of several lipophilic cations by reconstituted cytochromec oxidase vesicles and by similar vesicles exhibiting a potassium ion diffusion potential. While turnover of the oxidase generates a membrane potential of comparable magnitude to the potassium ion diffusion system, it is associated with a proton gradient of opposite polarity (interior basic). Both systems show rapid uptake of the permanently charged lipophilic cation, tetraphenylphosphonium, but only the potassium ion diffusion system accumulates the lipophilic amines doxorubicin and propranolol. This provides compelling evidence that such weak bases redistribute only in response to pH gradients and not membrane potential.     

Effects of potassium ions on proton motive force in Rhodobacter sphaeroides.

The proton motive force (PMF) was determined in Rhodobacter sphaeroides under anaerobic conditions in the dark and under aerobic-dark and anaerobic-light conditions. Anaerobically in the dark in potassium phosphate buffer, the PMF at pH 6 was -20 mV and was composed of an electrical potential (delta psi) only. At pH 7.9 the PMF was composed of a high delta psi of -98 mV and was partially compensated by a reversed pH gradient (delta pH) of +37 mV. ATPase inhibitors did not affect the delta psi, which was most likely the result of a K+ diffusion potential. Under energized conditions in the presence of K+ the delta psi depolarized due to electrogenic K+ uptake. This led to the generation of a delta pH (inside alkaline) in the external pH range of 6 to 8. This delta pH was dependent on the K+ concentration and was maximal at external K+ concentrations larger than 1.2 mM. In energized cells in 50 mM KPi buffer containing 5 mM MgSO4, a delta pH (inside alkaline) was present at external pHs from pH 6 to 8. As a result the overall magnitude of the PMF at various external pHs remained constant at -130 mV, which was significantly higher than the PMF under anaerobic-dark conditions. In the absence of K+, in 50 mM NaPi buffer containing 5 mM MgSO4, no depolarization of the delta psi was found and the PMF was composed of a large delta psi and a small delta pH. The delta pH became even reversed (inside acidic) at alkaline pHs (pH>7.3), resulting in a lowering of the PMF. These results demonstrate that in R. sphaeroides K+ uptake is essential for the generation of a delta pH and plays a central role in the regulation of the internal pH.     

Proton permeability of the plasma membrane of rat cortical synaptosomes

1. Transmembrane pH gradients (acidic inside) and electrical gradients (negative inside) were estimated in cortical synaptosomes from the distribution of the weak base methylamine and the lipophilic cation tetraphenylphosphonium, respectively. 2. Acidic interior pH gradients were produced by outwardly directed K+ gradients in Na+-free media. External K+ accelerated the dissipation of preformed H+ gradients. The appearance of H+ in the medium was directly demonstrated by pH-stat titration of a weakly buffered medium. Amiloride failed to inhibit K+-induced H+ release. 3. Elevating K+ in the absence of Na+ did not affect the endogenous contents of noradrenaline, dopamine, and serotonin, as determined by high-performance liquid chromatography with electrochemical detection. 4. H+ diffusion potentials were generated when outwardly directed H+ gradients were imposed onto the plasma membrane indicating an electrogenic H+ efflux which is not coupled to other ions. 5. At low K+ in the Na+-free sucrose medium, the plasma membrane potential Em (derived from distribution of tetraphenylphosphorium cation) did not approach a value for EK, the K+ equilibrium potential (calculated from K+ gradients). The deviation of Em from EK could be quantitatively described by a modified constant-field equation, taking a relative H+/K+ permeability coefficient of 12,400 into consideration. 6. It is concluded that synaptosomes have a H+ conductance pathway in their plasma membrane in addition to the Na+/H+ antiporter. H+ influx is driven by and leads to a reduction of Em. K+/H+ exchange resulted from the electrical coupling of K+ and H+ fluxes via parallel K+ and H+ channels. Since the Na+/H+ antiporter counteracts passive equilibration of H+ under physiological conditions, a continuous cycling of H+ across the plasma membrane will take place. A possible physiological role of the H+ leak in pHi regulation is discussed.     

Roles of K+ and Na+ in pH homeostasis and growth of the marine bacterium Vibrio alginolyticus.

The marine bacterium Vibrio alginolyticus, containing 470 mM-K+ and 70 mM-Na+ inside its cells, was able to regulate the cytoplasmic pH (pH(in)) in the narrow range 7.6-7.8 over the external pH (pH(out)) range 6.0-9.0 in the presence of 400 mM-Na+ and 10 mM-K+. In the absence of external K+, however, pHin was regulated only at alkaline pH(out) values above 7.6. When the cells were incubated in the presence of unusually high K+ (400 mM) and 4 mM Na+, the pH(in) was regulated only at acidic pH(out) values below 7.6. These results could be explained by postulating a K+/H+ antiporter as the regulator of pH(in) over the pH(out) range 6.0-9.0. When Na(+)-loaded/K(+)-depleted cells were incubated in 400 mM-Na+ in the absence of K+, an inside acidic delta pH was generated at pH(out) values above 7.0. After addition of diethanolamine the inside acidic delta pH collapsed transiently and then returned to the original value concomitant with the extrusion of Na+, suggesting the participation of a Na+/H+ antiporter for the generation of an inside acidic delta pH. In the presence of 400 mM-K+, at least 5 mM-Na+ was required to support cell growth at pH(out) below 7.5. An increase in Na+ concentration allowed the cells to grow at a more alkaline pH(out). Furthermore, cells containing more Na+ inside could more easily adapt to grow at alkaline pH(out). These results indicated the importance of Na+ in acidification of the cell interior via a Na+/H+ antiporter in order to support cell growth at alkaline pH(out) under conditions where the activity of a K+/H+ antiporter is marginal.     

Ion-dependent generation of the electrochemical proton gradient delta muH+ in reconstituted plasma membrane vesicles from the yeast Metschnikowia reukaufii

Plasma membrane vesicles were reconstituted by freezing and thawing of purified plasma membrane fraction from the yeast Metschnikowia reukaufii and phosphatidylcholine (type II-S from Sigma). The reconstituted plasma membrane vesicles generated a proton gradient (acidic inside) upon addition of ATP in presence of alkali cations. delta pH generation was most efficient when K+ was present both outside and inside the plasma membrane vesicles. Both ATPase activity and proton translocation in plasma membrane vesicles were inhibited by orthovanadate (50% inhibition at 100 microM). Plasma membrane vesicles reconstituted without added phosphatidylcholine generated in addition to delta pH, also an electrical potential difference delta psi (inside positive). Delta psi generation exhibited no K+ specificity. 50 microM dicyclohexylcarbodiimide inhibited completely delta psi generation whereas the K+-channel blocker quinine (5 microM) caused an 8-fold increase of delta psi. The proton gradient was much less affected by the agents. Taking into account the K+-dependent stimulation of the plasma membrane ATPase of M. reukaufii, these results further support the conclusion that the ATPase operates as a partially electrogenic H+/K+ exchanger, as was also suggested for other yeast plasma membrane ATPases.     

The use of fluorescamine as a permeant probe to localize phosphatidylethanolamine in intact friend erythroleukaemic cells

The marine bacterium, Vibrio alginolyticus, regulates the cytoplasmic pH at about 7.8 over the pH range 6.0-9.0. By the addition of diethanolamine (a membrane-permeable amine) at pH 9.0, the internal pH was alkalized and simultaneously the cellular K+ was released. Following the K+ exit, the internal pH was acidified until 7.8, where the K+ exit leveled off. The K+ exit was mediated by a K+/H+ antiporter that is driven by the outwardly directed K+ gradient and ceases to function at the internal pH of 7.8 and below. The Na+-loaded cells assayed in the absence of KCl generated inside acidic delta pH at alkaline pH due to the function of an Na+/H+ antiporter, but the internal pH was not maintained at a constant value. At acidic pH range, the addition of KCl to the external medium was necessary for the alkalization of cell interior. These results suggested that in cooperation with the K+ uptake system and H+ pumps, the K+/H+ antiporter functions as a regulator of cytoplasmic pH to maintain a constant value of 7.8 over the pH range 6.0-9.0.     

Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp.

The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of delta psi (membrane potential, interior negative) and/or delta pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed delta psi increased the Na+ efflux rate (V) linearly, and the slope of V versus delta psi was higher at pH 9 than at pH 8. Kinetic experiments indicated that the delta psi caused a pronounced increase in the Vmax for Na+ efflux, whereas the Km values for Na+ were unaffected by the delta psi. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction. These results have also been observed in delta pH-driven Na+ efflux experiments. When Na(+)-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence. The results of H+ influx experiments showed a good coincidence with those of Na+ efflux. H+ influx was enhanced by an increase of delta psi or internal Na+ concentration and inhibited by high internal H+ concentration. These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.     

Hydrogenosomes in the rumen protozoon Dasytricha ruminantium Schuberg.

The vacuo-lysosomes of Hevea brasiliensis (rubber tree) constitute a suitable model system for the study of active transport and energization at the level of the membrane of plant vacuoles. The pH gradient (delta pH) and the membrane potential (delta psi) of vacuo-lysosomes were determined by means of the weak base methylamine and the lipophilic cation tetraphenylphosphonium. The values obtained depended strongly on the experimental conditions such as medium pH or K+ concentration. Under experimental conditions, i.e., pH 7.5 outside and low K+, the delta pH amounts to about 0.9 unit, interior acid, and the delta psi to -120 mV, interior negative. The delta psi is presumably caused by the imposed K+ gradient, and the internal acidification might be a consequence of the passive proton inflow along the electric field. This explanation is sustained by the ineffectiveness of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in destroying the delta pH and delta psi, whereas higher K+ concentration decreased both. Under conditions existing in vivo, the membrane potential might be significantly lower. The presence of ATP increased the acidification of the intravesicular space by 0.5pH unit to a delta pH of up to 1.4 and shifts the membrane potential at least 60mV to a more positive value. The change of the protonmotive potential did not occur with ADP; the pH-dependence of the change was identical with the pH-dependence of a vacuo-lysosomal membrane-bound ATPase, and the effect of ATPase was prevented by the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The change of protonmotive potential difference, brought about by the ATPase, was at least 90 mV. This is evidence that a vacuo-lysosomal ATPase in plants can function as an electrogenic proton pump that transfers protons into the vacuo-lysosomal space.     

Uptake of basic amino acids and peptides into liposomes in response to transmembrane pH gradients.

The uptake of derivatives of lysine and a pentapeptide (ala-met-leu-trp-ala) into large unilamellar vesicle (LUV) systems in response to transmembrane pH gradients has been examined. In these derivatives, the C-terminal carboxyl functions have been converted to methyl esters or amides. It is shown that the presence of a pH gradient (interior acidic) results in the rapid and efficient accumulation of these weak base amino acid and peptide derivatives into LUVs in a manner consistent with permeation of the neutral (deprotonated) form. It is suggested that this property may have general implications for mechanisms of transbilayer translocation of peptides, such as signal sequences, which exhibit weak base characteristics.     

Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.     

Biological amine transport in chromaffin ghosts. Coupling to the transmembrane proton and potential gradients.

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in chromaffin ghosts. The chromaffin ghosts were formed by hypo-osmotic lysis of isolated bovine chromaffin granules and extensive dialysis in order to remove intragranular binding components and dissipate the endogenous electrochemical gradients. Upon ATP addition to suspensions of chromaffin ghosts, a transmembrane proton gradient alone, a transmembrane gradient alone, or both, could be established, depending upon the compositions of the media in which the ghosts were formed and resuspended. When chloride was present in the medium, addition of ATP resulted in the generation of a transmembrane proton gradient, acidic inside of 1 pH unit (measured by [14C]methylamine distribution), and no transmembrane potential (measured by [14C]-thiocyanate distribution). When ATP was added to chromaffin ghosts suspended in a medium in which chloride was substituted by isethionate, a transmembrane potential, inside positive, of 45 mV and no transmembrane proton gradient, was measured. In each medium, the addition of agents known to affect proton or potential gradients, respectively, exerted a predictable mechanism of action. Accumulation of [14C]epinephrine or [14C]5-hydroxytryptamine was over 1 order of magnitude greater in the presence of the transmembrane proton gradient or the transmembrane potential than in the absence of any gradient and, moreover, was related to the magnitude of the proton or potential gradient in a dose-dependent manner. When ghosts were added to a medium containing chloride and isethionate, both a delta pH and delta psi could be generated upon addition of ATP. In this preparation, the maximal rate of amine accumulation was observed. The results indicate that amine accumulation into chromaffin ghosts can occur in the presence of either a transmembrane proton gradient, or a transmembrane potential gradient, and that the maximal rate of accumulation may exist when both components of the protonmotive force are present.     

Anomalous driving force for renal brush border H+/OH-transport characterized by using 6-carboxyfluorescein

The pH, delta pH, and membrane potential dependences of H+/OH-permeability in renal brush border membrane vesicles (BBMV) were studied by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Quantitative H+/OH-fluxes (JH) were obtained from a calibration of the fluorescence response of 6CF to intravesicular pH using vesicles prepared with varying intravesicular and solution pHs. Intravesicular buffer capacity, determined by titration of lysed vesicles, increased monotonically from 140 to 260 mequiv/L in the pH range 5-8. JH was measured by subjecting voltage-clamped BBMV (K+/valinomycin) to preformed pH gradients over the pH range 5-8 and measuring the rate of change of intravesicular pH. For small preformed pH gradients (0.4 pH unit) JH [6 nequiv s-1 (mg of protein)-1] was nearly independent of pH (5-8), predicting a highly pH dependent H+ permeability coefficient. JH increased in a curvilinear manner from 6 to 104 nequiv s-1 (mg of protein)-1 as delta pH increased from 0.4 to 2.5. JH increased linearly [1.6-7.3 nequiv s-1 (mg of protein)-1] with induced K+ diffusion potentials (21-83 mV) in the absence of a pH gradient. These findings cannot be explained by simple diffusion of H+ or OH- or by mobile carrier models. Two mechanisms are proposed, including a lipid diffusion mechanism, facilitated by binding of H+/OH- to fixed sites in the membrane, and a linear H2O strand model, where dissociation of H2O in the membrane fixes H+ and OH- concentrations in strands, which can result in net H+/OH- transport.     

Electrochemical potential of protons in vesicles reconstituted from purified, proton-translocating adenosine triphosphatase.

Measurements were made of the difference in the electrochemical potential of protons (delta-mu H+) across the membrane of vesicles restituted from the ATPase complex (TF0.F1) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential (delta psi) and pH difference across the membrane (delta pH), respectively. In the presence of Tris buffer the maximal delta psi ans no delta pH were produced, while in the presence of the permeant anion NO-3 the maximal delta pH and a low delta psi were produced by the addition of ATP. When thATP concentration was 0.24 mm, the delta psi was 140-150 mV (positive inside) in Tris buffer, and the delta pH was 2.9-3.5 units (acidic inside) in the presence of NO-3. Addition of a saturating amount of ATP produced somewhat larger delta psi and delta pH values, and the delta -muH+attained was about 310mV. By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4-5 during ATP hydrolysis. The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.     

Existence of an adenosine 5'-triphosphate dependent proton translocase in bovine neurosecretory granule membrane

The addition of ATP to bovine neurohypophysial secretory granules suspended in isotonic sucrose medium induces a positive polarization, delta psi, of their interior without affecting their internal pH. In KCl-containing media, ATP failed to generate large delta psi but induced a pH gradient (delta pH; interior acidic). These observations are consistent with the existence in the neurosecretory granule membrane of an ATP-dependent inward electrogenic H+ translocase (H+ pump), capable in KCl-containing media of acidifying the granule matrix by H+-Cl- cotransport. The delta psi and delta pH generated by the H+ pump, defined as the ATP-induced changes sensitive to the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), were blocked by N,N'-dicyclohexylcarbodiimide, an inhibitor of all H+ pumps, and were insensitive to oligomycin, a mitochondrial ATPase inhibitor. In sucrose medium, measurements were complicated by a Donnan equilibrium reflecting the presence in the granule of peptide hormones and neurophysins which resulted in a CCCP-resistant resting delta pH. In KCl-containing media, the Donnan equilibrium was destroyed since the membrane is permeable to cations, but under these conditions a CCCP-resistant K+-diffusion potential was observed. The ATP-induced delta psi was also monitored by the extrinsic fluorescent probe bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol. The hypothesis of a granule H+ pump is further supported by the presence of an oligomycin-resistant ATPase in the preparation and the ultrastructural localization of such an activity on the granule membrane. The H+ pump has been found in both newly formed and aged neurosecretory granules. Its possible physiological function is discussed with reference to that of chromaffin granules, with which it has many similarities.     

The lysosomal proton pump is electrogenic

Lysosomes were purified approximately 40-fold from rat kidney cortex by differential and Percoll density gradient centrifugation. In a sucrose medium, the lysosomes quenched the fluorescence of the potential sensitive dye diS-C3-(5) (3,3'-dipropylthiocarbo-cyanine iodide) in a time-dependent manner, indicating that the dye accumulates within the lysosomal interior. After treatment of the lysosomes with valinomycin, the dye fluorescence displayed a logarithmic dependence upon the external K+ concentration; thus, the fluorescence signal provides a semiquantitative measure of the lysosomal membrane potential (delta psi). In the absence of valinomycin, lysosomal quenching of diS-C3-(5) fluorescence was partially reversed by agents which collapse the lysosomal pH gradient (ammonium sulfate, chloroquine, and K nigericin), suggesting that the proton gradient across the lysosomal membrane contributes to delta psi. A rapid increase in diS-C3-(5) fluorescence, indicative of an increase in delta psi, was observed upon the addition of Mg-ATP to the lysosomes. The ATP-dependent fluorescence change was inhibited by protonophores, K valinomycin, permeable anions, and N-ethylmaleimide, but was unaffected by ammonium sulfate, K nigericin, or sodium vanadate. Oligomycin had no effect at concentrations below 2 micrograms/ml; at higher concentrations, oligomycin partially inhibited the fluorescence response to Mg-ATP, but it also inhibited the fluorescence response to K valinomycin, suggesting that it had modified the permeability of the lysosomal membrane. Dicylohexylcarbodiimide behaved similarly to oligomycin. Mg-ATP also altered the lysosomal distribution of 86Rb+ (in the presence of valinomycin) and S[14C]CN-, consistent with an increase in the potential of the lysosomal interior of 40-50 mV. The results demonstrate that the lysosomal proton pump is electrogenic.     

Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient

Previous work has shown that adriamycin can be accumulated into large unilamellar vesicle (LUV) systems in response to K⁺ diffusion potential established by valinomycin. It is demonstrated here that adriamycin can also be rapidly and efficiently accumulated into egg phosphatidylcholine (egg PC) and egg PC-cholesterol (1:1) LUVs in response to a transmembrane pH gradient (interior acidic) in the absence of ionophores. This ‘active’ loading gives rise to trapping efficiencies as high as 98%, interior drug concentrations as high as 100 mM and significantly enhances drug retention within the vesicles. This procedure may be of general utility for loading liposomal systems for in vivo drug delivery.