Ca(2+)-induced fusion of phospholipid vesicles containing free fatty acids: modulation by transmembrane pH gradients.

The influence of a transmembrane pH gradient on the Ca(2+)-induced fusion of phospholipid vesicles, containing free fatty acids, has been investigated. Large unilamellar vesicles composed of an equimolar mixture of cardiolipin, dioleoylphosphatidylcholine, and cholesterol, containing 20 mol % oleic acid, were employed. Fusion was measured using a kinetic assay for lipid mixing, based on fluorescence resonance energy transfer. At pH 7.5, but not at pH 6.0, in the absence of a pH gradient, oleic acid stimulates the fusion of the vesicles by shifting the Ca2+ threshold concentration required for aggregation and fusion of the vesicles from about 13 mM to 10 mM. In the presence of a pH gradient (at an external pH of 7.5 and a vesicle interior pH of 10.5), the vesicles exhibit fusion characteristics similar to vesicles that do not contain oleic acid at all, consistent with an effective sequestration of the fatty acid to the inner monolayer of the vesicle bilayer induced by the imposed pH gradient. The kinetics of the fusion process upon simultaneous generation of the pH gradient across the vesicle bilayer and initiation of the fusion reaction show that the inward movement of oleic acid in response to the pH gradient is extremely fast, occurring well within 1 s. Conversely, dissipation of an imposed pH gradient, by addition of a proton ionophore during the course of the fusion process, results in a rapid enhancement of the rate of fusion due to reequilibration of the oleic acid between the two bilayers leaflets.     

Lipid asymmetry induced by transmembrane pH gradients in large unilamellar vesicles

We have investigated the influence of transmembrane pH gradients across large unilamellar vesicle membranes on the transbilayer distributions of simple lipids with weak base and weak acid characteristics. Trinitrobenzenesulfonic acid labeling results consistent with a rapid and complete migration of stearylamine and sphingosine to the inner monolayer of the large unilamellar vesicles are observed when the large unilamellar vesicles' interior is acidic. Alternatively, when the vesicle interior is basic, oleic and stearic acid cannot be removed by external bovine serum albumin, indicating a localization in the inner monolayer. Moreover, effects corresponding to the decrease in external surface charge predicted upon the migration of stearylamine or stearic acid to the inner monolayer are readily detected employing ion exchange chromatography. These results are consistent with transbilayer distributions of these agents dictated by a Henderson-Hasselbach equilibrium. The possible implications for metabolic regulation by pH gradients, as well as factors giving rise to phospholipid transbilayer asymmetry, are discussed.     

Use of the fluorescent weak acid dansylglycine to measure transmembrane proton concentration gradients

The amphiphilic fluorescent dye N-[(5-dimethylamino)naphth-1-ylsulfonyl]glycine (dansylglycine) can be used to monitor the magnitude and stability of transmembrane proton gradients. Although freely soluble in aqueous media, the dye readily adsorbs to the surfaces of lipid vesicles. Because membrane-bound dye fluoresces at a higher frequency, and with greater efficiency, than dye in aqueous solution, it is easy to isolate the fluorescence emission from those dye molecules adsorbed to the lipid surface. When dansylglycine is mixed with phospholipid vesicles, the dye molecules attain a partition equilibrium between buffer and the outer, proximal surface of the vesicles. This is a rapid, diffusion-limited process that is indicated by a fast phase of fluorescence intensity increase monitored at 510 nm. In a second step, the inner, distal surface of each vesicle becomes populated with dye, a process that involves permeation through the lipid bilayer and that is generally much slower than the original adsorption step. Dansylglycine is a weak acid that permeates as an electrically neutral species; the flux of dye across the bilayer is thus strongly dependent on the degree of protonation of the dye's carboxylate moiety. When the external pH is lower than that of the vesicle lumen, the inward flux of dye is greater than that in the opposite direction, and dye accumulates in the lumen. This leads to a local elevation of dansylglycine concentration in the inner membrane monolayer, which in turn results in an elevated fluorescence intensity proportional to the membrane pH gradient.     

Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients

The influence of membrane pH gradients on the transbilayer distribution of some common phospholipids has been investigated. We demonstrate that the transbilayer equilibrium of the acidic phospholipids egg phosphatidylglycerol (EPG) and egg phosphatidic acid (EPA) can be manipulated by membrane proton gradients, whereas phosphatidylethanolamine, a zwitterionic phospholipid, remains equally distributed between the inner and outer monolayers of large unilamellar vesicles (LUVs). Asymmetry of EPG is examined in detail and demonstrated by employing three independent techniques: ion-exchange chromatography, 13C NMR, and periodic acid oxidation of the (exterior) EPG headgroup. In the absence of a transmembrane pH gradient (delta pH) EPG is equally distributed between the outer and inner monolayers of LUVs. When vesicles composed of either egg phosphatidylcholine (EPC) or DOPC together with 5 mol % EPG are prepared with a transmembrane delta pH (inside basic, outside acidic), EPG equilibrates across the bilayer until 80-90% of the EPG is located in the inner monolayer. Reversing the pH gradient (inside acidic, outside basic) results in the opposite asymmetry. The rate at which EPG equilibrates across the membrane is temperature dependent. These observations are consistent with a mechanism in which the protonated (neutral) species of EPG is able to traverse the bilayer. Under these circumstances EPG would be expected to equilibrate across the bilayer in a manner that reflects the transmembrane proton gradient. A similar mechanism has been demonstrated to apply to simple lipids that exhibit weak acid or base characteristics [Hope, M. J., & Cullis, P. R. (1987) J. Biol. Chem 262, 4360-4366]     

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.     

Detection of surface charge-related properties in model membrane systems by aqueous two-phase partition

Unilamellar vesicles composed of phosphatidylcholine (PC) and either phosphatidic acid (PA) or phosphatidylglycerol (PG) partition to the upper poly(ethylene glycol) (PEG)-rich phase of a charge-sensitive 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 7, consistent with the vesicles bearing a net negative charge. When prepared in the presence of a pH gradient (interior acidic), PC/PA vesicles exhibit an increased partition to the top PEG-rich phase, consistent with a redistribution of the PA from the inner to the outer monolayer of the vesicle bilayer. Conversely, when prepared in the presence of a pH gradient (interior basic), PC/PG vesicles exhibit a decreased top-phase partition, consistent with a redistribution of the PG from the outer to the inner monolayer of the vesicle bilayer. Unilamellar vesicles composed of PC and stearylamine partition to the lower dextran-rich phase of a 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 8.5, consistent with the vesicles bearing a net positive charge. When prepared in the presence of a pH gradient (interior acidic), conditions under which the stearylamine is trapped on the inner monolayer of the bilayer, the vesicles now partition predominantly to the interface in a manner similar to vesicles composed of PC alone. These results demonstrate that partitioning in aqueous two-phase polymer systems is a sensitive method for monitoring the asymmetry of charged lipids in model membrane systems and also suggests that partitioning in charge-sensitive systems depends only on the physical nature of the exterior surface of the membrane.     

Determination of osmotic volumes and pH gradients of plant membrane and lipid vesicles using ESR spectroscopy

Volumes and pH gradients were determined with spin probes in liposomes and zucchini membrane vesicles by quantitating the internal concentrations of probes in the presence of an impermeable line-broadening agent, manganese + EDTA. Volume shrinkage in response to increasing external concentrations of MnEDTA was consistent with perfect osmotic behavior of both vesicle populations. Buffer additions were used to impose pH gradients on the vesicles; liposome gradients measured with a spin-labeled weak acid were slightly smaller than the maximum theoretical imposed gradients, whereas above a threshold magnitude, measured gradients for the plant membranes were significantly smaller than imposed gradients. However, the residual pH gradient in the zucchini vesicles decreased at about the same rate as the liposome gradient. Moreover, this residual gradient was not completely collapsed in the presence of the proton ionophore, FCCP, indicating that the vesicles were impermeable to ions; indeed, ion permeabilities of both vesicle preparations appeared to be similar during the slow phase of the pH gradient collapse. Thus, zucchini membrane vesicles are tightly sealed and appear to have a mechanism for dissipating pH gradients rapidly when these gradients exceed some threshold value.     

Aggregation, lipid exchange, and metastable phases of dimyristoylphosphatidylethanolamine vesicles

A new fluorescent lipid analogue, bimanephosphatidylcholine, has been synthesized for use in lipid bilayers. This probe is well suited as an energy-transfer donor with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine as the acceptor. Dimyristoylphosphatidylethanolamine vesicles are prepared by sonication at pH 9 and characterized by electron microscopy and other methods. Resonance energy transfer between separately labeled donor and acceptor vesicles is monitored during HCl-induced aggregation to determine the kinetics of lipid randomization. Light scattering is also monitored to measure the kinetics of aggregation. The light scattering shows a marked reversal with NaOH while the energy transfer does not, indicating lipid exchange during a reversibly aggregated state; the extent of energy transfer suggests that only lipids in the outer monolayers exchange. The gel to liquid-crystalline phase transition temperature in HCl-treated vesicles is found to be 47 degrees C with diphenylhexatriene. The initial sonicated dispersion does not show a sharp phase transition. In vesicles labeled with both donor and acceptor probes, a small, irreversible increase in energy transfer is obtained upon lowering and then restoring the pH. These results suggest a metastable phase in the sonicated vesicles containing a randomized distribution of lipid and probes within the bilayers; the thermodynamically favored phase, whose formation is triggered by the pH shock, contains domains within which the probe lipids are more highly concentrated.     

Trans-potassium effects on the chloride/proton symporter activity of guinea-pig ileal brush-border membrane vesicles.

To investigate the inhibitory effect of trans potassium on the Cl-/H+ symporter activity of brush-border membrane vesicles from guinea pig ileum, we measured both 36Cl uptake and, by the pyranine fluorescence method, proton fluxes, in the presence of appropriate H+ and K+ gradients. In the absence of valinomycin, a time-dependent inhibitory effect of chloride uptake by trans K+ was demonstrated. This inhibition was independent of the presence or absence of any K+ gradient. Electrical effects cannot be involved to explain these inhibitions because the intrinsic permeability of these vesicles to Cl- and K+ is negligibly small. Rather, our results show that, in the absence of valinomycin, the inhibitory effect of intravesicular K+ involves an acceleration of the rate of dissipation of the proton gradient through an electroneutral exchange of trans K+ for cis H+, catalyzed by the K+/H+ antiporter also present in these membranes. Valinomycin can further accelerate the rate of pH gradient dissipation by facilitating an electrically-coupled exchange between K+ and H+. To evaluate the apparent rate of pH-dissipating, downhill proton influx, we measured chloride uptake by vesicles preincubated in the presence of alkaline-inside pH gradients (pHout/pHin = 5.0/7.5), charged or not with K+. In the absence of intravesicular K+, proton influx exhibited monoexponential kinetics with a time constant k = 11 s-1. Presence of 100 mM K+ within the vesicles significantly increased the rate of pH gradient dissipation which, furthermore, became bi-exponential and revealed the appearance of an additional, faster proton influx component with k = 71 s-1. This new component we interpret as representing the sum of the electroneutral and the electrically-coupled exchange of trans K+ for cis H+, mentioned above. Finally, by using the pH-sensitive fluorophore, pyranine, we demonstrate that, independent of the absence or presence of a pH gradient, either vesicle acidification or alkalinisation can be generated by adding, respectively, Cl- or K+ to the extravesicular medium. Such results confirm the independent existence of both Cl-/H+ symporter and K+/H+ antiporter activities in our vesicle preparations, the relative activity of the former being larger under the conditions of the present experiments. The possible interplay of these two proton-transfer mechanisms in the regulation of the intracellular pH is discussed.     

Lipid vesicle-cell interactions. I. Hemagglutination and hemolysis

The interaction of lipid vesicles (liposomes) of several different compositions with erythrocytes has been investigated. Lecithin liposomes, rendered positively charged with stearylamine, exhibit potent hemagglutination activity in media containing low concentrations of electrolytes. The hemagglutination titer is found to be a linear function of the zeta potential of the lipid vesicles. Hemagglutination is reduced when the surface potential of the cells is made more positive by pH adjustment or enzyme treatment. Similarly, hemagglutination is reduced by increasing concentrations of electrolytes. Hemagglutination is examined theoretically and is shown to be consistent with vesicle-cell interactions that are due to only electrostatic forces. Vesicles containing lysolecithin in addition to lecithin and stearylamine cause lysis of erythrocytes, provided the lipids of the vesicles are above the crystal-liquid crystal phase transition temperature. In addition, hemolysis requires close juxtaposition of the vesicle to the cell membrane; vesicles precoated with antibodies exhibit severely diminished hemolytic activities, only a small fraction of which can be attributed to a reduction in hemagglutination titer. Evidence is presented indicating that a single vesicle is sufficient to lyse one cell. With regard to hemagglutination and hemolysis, lipid vesicles of simple composition mimic paramyxoviruses such as Sendai virus.     

Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters

Uptake of 22Na+ and 45Ca2+ into everted membrane vesicles from Escherichia coli was measured with imposed transmembrane pH gradients, acid interior, as driving force. Vesicles loaded with 0.5 M KCl were diluted into 0.5 M choline chloride to create a potassium gradient. Addition of nigericin to produce K+/H+ exchange resulted in formation of a pH gradient. This imposed gradient was capable of driving 45Ca2+ accumulation. In another method vesicles loaded with 0.5 M NH4Cl were diluted into 0.5 M choline chloride, creating an ammonium diffusion potential. A gradient of H+ was produced by passive efflux of NH3. With an ammonium gradient as driving force, everted vesicles accumulated both 45Ca2+ and 22Na+. The data suggest that 22Na+ uptake was via the sodium/proton antiporter and 45Ca2+ via the calcium/proton antiporter. Uptake of both cations required alkaline pHout. A minimum pH gradient of 0.9 unit was needed for transport of either ion, suggesting gating of the antiporters. Octyl glucoside extracts of inner membrane were reconstituted with E. coli phospholipids in 0.5 M NH4Cl. NH4+-loaded proteoliposomes accumulated both 22Na+ and 45Ca2+, demonstrating that the sodium/proton and calcium/proton antiporters could be solubilized and reconstituted in a functional form.     

Determination of transmembrane pH gradients and membrane potentials in liposomes.

Techniques for determining large transbilayer pH gradients (delta pH) and membrane potentials (delta psi) induced in response to delta pH in large unilamellar vesicle liposomal systems by measuring the transbilayer redistribution of radiolabeled compounds have been examined. For liposomes with acidic interiors, it is shown that protocols using radiolabeled methylamine in conjunction with gel filtration procedures to remove untrapped methylamine provide accurate measures of delta pH in most situations. Exceptions include gel state lipid systems, where transbilayer equilibration processes are slow, and situations where the interior buffering capacity is limited. These problems can be circumvented by incubation at elevated temperatures and by using probes with higher specific activities, respectively. Determination of delta pH in vesicles with a basic interior using weak acid probes such as radiolabeled acetate in conjunction with gel filtration was found to be less reliable, and an alternative equilibrium centrifugation protocol is described. In the case of determinations of the membrane potentials induced in response to these pH gradients, probes such as tetraphenylphosphonium and thiocyanate provide relatively accurate measures of the delta psi induced. It is shown that the maximum transmembrane pH gradient that can be stably maintained by an egg phosphatidylcholine-cholesterol 100-nm-diam large unilamellar vesicle is approximately 3.7 units, corresponding to an induced delta psi of 220 mV or transbilayer electrical field of 5 x 10(5) V/cm.     

Phosphatidylcholine-fatty acid membranes. I. Effects of protonation, salt concentration, temperature and chain-length on the colloidal and phase properties of mixed vesicles, bilayers and nonlamellar structures

The phase and colloidal properties of phosphatidylcholine/fatty acid (PC/FA) mixed vesicles have been investigated by optical methods, acid-base titration, and theoretically as a function of temperature (5-80 degrees C), molar lipid ratio (0-1), lipid chain length (C14-C18), headgroup ionization (1.5 less than or equal to pH less than or equal to 10), vesicle concentration (0.05-32 mumol vesicle.dm-3, and ionic strength (0.005 less than or equal to J less than or equal to 0.25). Increasing the fatty acid concentration in PC bilayers causes the phase transition temperatures (at 4 less than or equal to pH less than or equal to 5) to rise until, for more than 2 FA molecules per PC molecule, the sample turbidity exhibits only two transitions corresponding to the chain-melting of the 1:2 stoichiometric complexes of PC/FA, and pure fatty acid. The former transition is into a nonlamellar phase and is accompanied by extremely rapid vesicle aggregation (with association rates on the order of Ca approximately 10(7) dm3.mol-1.s-1) and massive lipid precipitation. Fluid-phase vesicles with less than 2 FA per PC associate much more slowly (Ca approximately 10(3) dm3.mol-1.s-1), their aggregation being comparable to that of the ordered-phase liposomes. Under no conditions was the relation between the fatty acid concentration and the vesicle association rate for the fluid-phase vesicles linear. In contrast to the X-ray diffraction data, optical measurements reveal a 'pretransitional region' between the chain-melting temperature of the PC component and the temperature at which the gross transformation into a nonlamellar phase sets in. This is seen for all lipid mixtures investigated. On the relative temperature scale, lipids with different chain lengths behave qualitatively similarly; however, the effective association constants determined for samples of constant lipid concentration seem to decrease somewhat with the number of CH2 groups per chain. Fatty acid protonation, which yields electrically neutral bilayers, invariably increases the rate of vesicle association; we have measured, for example, Ca approximately 10(2) at pH approximately 7 and Ca approximately 10(7) dm3.mol-1.s-1 at pH approximately 4). Protonation of the phosphatidylcholine phosphate groups, which causes a net positive charge to accumulate on the lipid vesicles, initially increases (Ca approximately 10(8) dm3.mol-1.s-1) but ultimately decreases (Ca approximately 10(7) dm3.mol-1.s-1) the rate of association between PC/FA (1:2) mixed vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nitrite Transport in Chloroplast Inner Envelope Vesicles (I. Direct Measurement of Proton-Linked Transport)

Chloroplast inner envelope membrane vesicles that are loaded with the pH-sensitive fluorophore, pyranine, show rapid internal acidification when nitrite is added. Acidification is dependent upon [delta]pH, with the inside of vesicles being alkaline with respect to the outside. The rate of vesicle acidification was directly proportional to the concentration of nitrite that was added and the imposed pH difference across the membrane. In contrast, added nitrate had no effect on vesicle acidification. Nitrite also caused acidification of asolectin vesicles. The extent of vesicle acidification is dependent on the internal volume of vesicles. Inner envelope and asolectin vesicles that were prepared by extrusion were approximately the same size, allowing them to be compared when the final extent of acidification, measured after the pH gradient had collapsed, was similar. The rate of nitrite-dependent acidification was similar in these two preparations at any single nitrite concentration. These results indicate that nitrite movement occurs by rapid diffusion across membranes as nitrous acid, and this movement is dependent on a proton gradient across the lipid bilayer. Under conditions approximating those in vivo, the rate of diffusion of nitrous acid far exceeds that of nitrite reduction within chloroplasts.     

A novel method for the efficient entrapment of calcium in large unilamellar phospholipid vesicles

A technique for the efficient entrapment of high concentrations of Ca2+ in large unilamellar phospholipid vesicles (LUVs), using the carboxylic acid antibiotic ionophore A23187 (calcimycin) is demonstrated. It is shown that rapid A23187-mediated entrapment of Ca2+, corresponding to essentially 100% sequestration of the extravesicular cation may be achieved for egg yolk phosphatidylcholine LUVs (100 nm) in the presence of a transmembrane proton gradient (acidic interior). Interior-exterior concentration cation gradients of over 400-fold may be readily achieved, with interior Ca2+ concentrations in excess of 250 mM. It is shown that the extent and efficiency of the A23187-mediated uptake process is affected by the intravesicular buffering capacity and the extravesicular Ca2+ concentration in a manner that is consistent with a Ca2(+)-H+ exchange process. In the absence of a pH gradient, or the presence of a reversed gradient (basic interior), only background levels of cation uptake are detected. The driving force for A23187-mediated uptake of Ca2+ is shown to depend on the intravesicular proton pool rather than on a chelation process. This protocol provides a novel method for the efficient entrapment of high concentrations of Ca2+ and other cations in phospholipid vesicles.     

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.     

Hydroxyl/bile acid exchange. A new mechanism for the uphill transport of cholate by basolateral liver plasma membrane vesicles

In order to characterize the driving forces for the concentrative uptake of unconjugated bile acids by the hepatocyte, the effects of pH gradients on the uptake of [3H]cholate by rat basolateral liver plasma membrane vesicles were studied. In the presence of an outwardly directed hydroxyl gradient (pH 6.0 outside and pH 7.5 inside the vesicle), cholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than at equilibrium ("overshoot"). In the absence of a pH gradient (pH 6.0 or 7.5 both inside and outside the vesicle), uptake was relatively slower and no overshoot was seen. Reductions in the magnitude of the transmembrane pH gradient were associated with slower initial uptake rates and smaller overshoots. Cholate uptake under pH gradient conditions was inhibited by furosemide and bumetanide but not by 4, 4'-diisothiocyano-2,2'-disulfonic stilbene (SITS), 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (DIDS), or probenecid. In the absence of a pH gradient, an inside-positive valinomycin-induced K+ diffusion potential caused a slight increase in cholate uptake which was insensitive to furosemide. Moreover, in the presence of an outwardly directed hydroxyl gradient, uphill cholate transport was observed even under voltage clamped conditions. These findings suggest that pH gradient-driven cholate uptake was not due to associated electrical potentials. Despite an identical pKa to that of cholate, an outwardly directed hydroxyl gradient did not drive uphill transport of three other unconjugated bile acids (deoxycholate, chenodeoxycholate, ursodeoxycholate), suggesting that a non-ionic diffusion mechanism cannot account for uphill cholate transport. In canalicular vesicles, although cholate uptake was relatively faster in the presence of a pH gradient than in the absence of a gradient, peak uptake was only slightly above that found at equilibrium under voltage clamped conditions. These findings suggest a specific carrier on the basolateral membrane of the hepatocyte which mediates hydroxyl/cholate exchange (or H+-cholate co-transport). A model for uphill cholate transport is discussed in which the Na+ pump would ultimately drive Na+/H+ exchange which in turn would drive hydroxyl/cholate exchange.     

Promotion of acid-induced membrane fusion by basic peptides. Amino acid and phospholipid specificities

The ability of oligo- and polymers of the basic amino acids L-lysine, L-arginine, L-histidine and L-ornithine to induce lipid intermixing and membrane fusion among vesicles containing various anionic phospholipids has been investigated. Among vesicle consisting of either phosphatidylinositol or mixtures of phosphatidic acid and phosphatidylethanolamine rapid and extensive lipid intermixing, but not complete fusion, was induced at neutral pH by poly-L-ornithine or L-lysine peptides of five or more residues. When phosphatidylcholine was included in the vesicles, the lipid intermixing was severely inhibited. Such lipid intermixing was also much less pronounced among phosphatidylserine vesicles. Poly-L-arginine provoked considerable leakage from the various anionic vesicles and caused significantly less lipid intermixing than L-lysine peptides at neutral pH. When the addition of basic amino acid polymer was followed by acidification to pH 5-6, vesicle fusion was induced. Fusion was more pronounced among vesicles containing phosphatidylserine or phosphatidic acid than among those containing phosphatidylinositol, and occurred also with vesicles whose composition resembles that of cellular membranes (i.e., phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine, 50:30:20, by mol). Liposomes with this composition are resistant to fusion by Ca2+ or by acidification after lectin-mediated contact. The tight interaction among vesicles at neutral pH, resulting in lipid intermixing, does not seem to be necessary for the fusion occurring after acidification, but the basic peptides nevertheless appear to play a more active role in the fusion process than simply bringing the vesicles in contact. However, protonation of the polymer side chains and transformation of the polymer into a polycation does not explain the need for acidification, since the pH-dependence was quite similar for poly(L-histidine)- and poly(L-lysine)-mediated fusion.     

Isolation and reconstitution of the clathrin-coated vesicle proton translocating complex

Clathrin-coated vesicles contain a proton translocating ATPase which is insensitive to azide but inhibited by N-ethylmaleimide. The ATP hydrolytic subunit of this proton pump has been solubilized, partially purified, and reconstituted into H+-ATPase-depleted coated vesicle membranes (Xie, X.-S., Stone, D.K., and Racker, E. (1984) J. Biol. Chem. 259, 11676-11678). In this communication we report that the entire proton transporting complex has been solubilized and purified 200-fold. The complex, when reconstituted into brain lipid liposomes, catalyzes azide-resistant, N-ethylmaleimide-sensitive H+ transport manifested as both generation of a pH gradient and an electrical gradient. The complex has an apparent molecular mass of 530 kDa.     

Change in Ca2+ transport in myometrial plasma cell membrane in proton gradient dissipation

By means of delta pH 14C-methylamine indicator the myometrium vesicle sarcolemma fraction was shown to be capable, while applying a "delta pH-leap", for developing in it a proton transmembrane gradient, dissipating in time. The proton gradient dissipation under Ca ions transmembrane equilibrium concentration is a driving force of these ions transposition against the concentration gradient. The blocking agents of H+ transport--Cd ions and DCCD decrease the proton-dependent 45Ca2+ accumulation in the vesicle sarcolemma fraction. The conclusion has been made about the possibility of Ca2+(H(+)-exchange on the uterus smooth cells sarcolemma. The possible physiological value of this exchange is under discussion.