Cholesterol Stimulation of Penetration of Unilamellar Liposomes by Hydrophobic Compounds.

The incorporation of cholesterol into unilamellar liposomes greatly increased the transmembranous movement of hydrophobic ionophores such as nigericin. In reconstituted liposomes containing rhodopsin as the only protein, the presence of cholesterol lowers by 10-fold or more the amount of negericin required to eliminate the light-driven proton gradient. These effects are seen both above and below the transition temperature of the phospholipid used for reconstitution. Cholesterol similarly increases the ability of A-23187, 1799, or NH4SCN to collapse the proton gradient of bacteriorhodopsin vesicles. Cholesterol also lowers the concentration of nigericin or valinomycin required for a rapid translocation of Rb+ into protein-free liposomes. It also lowers the concentration of A-23187 required for the release of Ca45 trapped in protein-free liposomes. In contrast to these observations and in confirmation of previous findings, we observed that cholesterol decreased the permeability of liposomes for glucose. Thus the effects of cholesterol on the permeability of the membrane vary with the chemical nature of the permeating compounds. We have also confirmed that in multilamellar liposomes cholesterol decreases the permeability of Rb+ in the presence of valinomycin. It therefore appears that the effect of cholesterol changes with the size and structural features of the model membranes.     

A mechanism of respiratory control: studies with proteoliposomes containing cytochrome oxidase and bacteriorhodopsin

Both beef heart cytochrome oxidase and bacteriorhodopsin of Halobacterium halobium were reconstituted into liposomes by the sonication-cholate dialysis method. The proteoliposomes showed the respiratory control ratio of 4.2, and steady-state illumination of the vesicles lead to the 2.7-fold stimulation of the oxidase activity in the absence of uncouplers. The light-stimulated state 4 respiration increased with light intensity, but light had no effect on the oxidase activity that had been relieved by addition of uncouplers. Proteoliposomes with the photosensitive oxidase activity were also obtained when cytochrome oxidase vesicles were fused with bacteriorhodopsin vesicles in the presence of calcium chloride, and the extent of photoactivation was maximally 1.4-fold. The light-induced respiratory release was observed even in the presence of valinomycin or nigericin, indicating that the oxidase activity was sensitive to both the membrane potential and the pH gradient. We propose as a mechanism of the respiratory control that the process of proton transport to the reaction center for water formation is the rate limiting step for the cytochrome oxidase activity.     

Light-induced leucine transport in Halobacterium halobium envelope vesicles: a chemiosmotic system.

Halabacterium halobium cell envelope vesicles accumulate L-[14-C]leucine during illumination, against a large concentration gradient. Leucine uptake requires Na-+ and is optimal in KCl-loaded vesicles resuspended in KCl-NaCl solution (1.5 M:1.5 M). Half-maximal transport is seen at 1 X 10-minus 6 M leucine. In the dark the accumulated leucine is rapidly and exponentially lost from the vesicles. The action spectrum and the light-intensity dependence indicate that the transport is related to the extrusion of protons, mediated by bacteriorhodopsin. Since light gives rise to both a pH gradient and an opposing transmembrane potential (interior negative), it wass responsible for providing the energy for leucine transport. The following results were obtained under illumination: (1) membrane-permeant cations and valinomycin or gramicidin greatly inhibited leucine transport without altering the pH gradient; (2) buffering both inside and outside the vesicles eliminated the pH gradient while enhancing leucine transport; (3) dicyclohexylcarbodiimide increased the pH gradient without affecting leucine transport; (4) arsenate did not inhibit leucine uptake. A diffusion potential, established by adding valinomycin to KCl-loaded vesicles, caused leucine influx in the dark. These results suggest that the leucine transport system is not coupled to ATP hydrolysis, and responds to the membrane potential rather than to the pH gradient. The Na-+ dependence of the transport and the observation that a small NaCl pulse causes transient leucine influx in the dark in KCl-loaded vesicles, resuspended in KCl, even in the presence of p-trifluoromethoxycarbonylcyanide phenylhydrazone or with buffering, suggest that the translocation of leucine is facilitated by symport with Na-+.     

Bacteriorhodopsin in liposomes. II. Experimental evidence in support of a theoretical model

In the preceding article equations describing relevant ion flows in illuminated suspensions of bacteriorhodopsin liposomes have been derived. Here these equations are subjected to experimental tests. Changes in permeability characteristics of the liposomal membrane are brought about by addition of specific ionophores and change of medium composition. Using light-driven proton uptake and electrochemical potential differences for protons across the membrane as observation parameters, ridig attempts to falsify the derived equations are unsuccessful.Agreement between equations and experimental results is established on the point of: (i) the antagonistic effect of valinomycin and nigericin on the two components of the proton-motive force, (ii) the time dependence of the changes in transmembrane electrical and chemical potential differences after the onset of illumination.In three independent experimental systems evidence was obtained for the correctness of the postulated dependence of the turnover rate of the photochemical cycle on back pressure by the transmembrane electrochemical potential difference for protons.     

Cholesterol stimulation of penetration of unilamellar liposomes by hydrophobic compounds

Summary The incorporation of cholesterol into unilamellar liposomes greatly increased the transmembranous movement of hydrophobic ionophores such as nigericin. In reconstituted liposomes containing rhodopsin as the only protein, the presence of cholesterol lowers by 10-fold or more the amount of nigericin required to eliminate the light-driven proton gradient. These effects are seen both above and below the transition temperature of the phospholipid used for reconstitution.Cholesterol similarly increases the ability of A-23187, 1799, or NH₄SCN to collapse the proton gradient of bacteriorhodopsin vesicles. Cholesterol also lowers the concentration of nigericin or valinomycin required for a rapid translocation of Rb⁺ into protein-free liposomes. It also lowers the concentration of A-23187 required for the release of Ca⁴⁵ trapped in protein-free liposomes. In contrast to these observations and in confirmation of previous findings, we observed that cholesterol decreased the permeability of liposomes for glucose. Thus the effects of cholesterol on the permeability of the membrane vary with the chemical nature of the permeating compounds. We have also confirmed that in multilamellar liposomes cholesterol decreases the permeability of Rb⁺ in the presence of valinomycin. It therefore appears that the effect of cholesterol changes with the size and structural features of the model membranes.     

Interference of electron donors in the measurement of the proton gradient and membrane potential by flow dialysis

The three most commonly used electron donors for flow dialysis measurements of membrane potential lead to the development of an apparent but artifactual membrane potential with the interior negative in the presence or absence of membrane vesicles. The same three electron donors used in flow dialysis determinations of delta pH in the presence or absence of membrane vesicles lead to the development of an apparent but artifactual delta pH with the interior acidic. These artifacts have been evaluated using two probes for membrane potential, namely, TPP+ and rubidium in the presence of valinomycin and for two probes of delta pH, namely, acetate and DMO. Measurements were made over a range of ionic strengths.     

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.     

The effect of trypsin treatment on the incorporation and energy-transducing properties of bacteriorhodopsin in liposomes

Bacteriorhodopsin and trypsin-modified bacteriorhodopsin have been reconstituted into liposomes by means of a low pH-sonication procedure. The incorporation of bacteriorhodopsin in these proteoliposomes is predominantly in the same direction as in vivo and the direction of proton pumping is from inside to outside the liposomes. The direction of proton translocation and electrical potential generation was studied as a function of the reconstitution pH. Light-dependent proton extrusion and generation of a Δp, interior negative and alkaline was observed at a reconstitution pH below 3.0 using bacteriorhodopsin, and at a pH below 3.5 using trypsin-modified bacteriorhodopsin. The shift in inflection point is explained in terms of differences between bacteriorhodopsin and trypsin-modified bacteriorhodopsin in a specific protein-phospholipid interaction which depends on the surface charge density of the cytoplasmic side of bacteriorhodopsin. The magnitude of the protonmotive force (Δp) generated by trypsin-modified bacteriorhodopsin in liposomes was quantitated. Illumination of the proteoliposomes resulted in the generation of a high Δp (135 mV, inside negative and alkaline), with a major contribution of the pH gradient. The ionophores nigericin and valinomycin induced, respectively, a compensatory interconversion of ΔpH into Δψ and vice versa. If no endogenous proton permeability of the membrane would exist, a protonmotive force could be generated of − 143 mV as electrical potential alone or − 162 mV as pH gradient alone.     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR, diffusion and entrapment analyses.

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1--0.4 micron with a mean diameter of 0.21 micron.     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR,diffusion and entrapment analyses

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1–0.4 μm with a mean diameter of 0.21 μm.     

Interleukin-2-induced small unilamellar vesicle coalescence

Recombinant human interleukin-2 (rhIL-2) was incorporated in liposomes for potential therapeutic applications using a novel process. In this process, rhIL-2 caused the formation of large, unique multilamellar vesicles (MLVs) from small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC). Vesicle coalescence occurred most rapidly at 19 degrees C, between the pre- and main phase transition temperatures of DMPC, and showed a dependence upon pH (pH <5.5), ionic strength (>50 mM) and the initial size of the unilamellar vesicles (相似文献   

The influence of a transmembrane pH gradient on protonation probabilities of bacteriorhodopsin: the structural basis of the back-pressure effect

Bacteriorhodopsin pumps protons across a membrane using the energy of light. The proton pumping is inhibited when the transmembrane proton gradient that the protein generates becomes larger than four pH units. This phenomenon is known as the back-pressure effect. Here, we investigate the structural basis of this effect by predicting the influence of a transmembrane pH gradient on the titration behavior of bacteriorhodopsin. For this purpose we introduce a method that accounts for a pH gradient in protonation probability calculations. The method considers that in a transmembrane protein, which is exposed to two different aqueous phases, each titratable residue is accessible for protons from one side of the membrane depending on its hydrogen-bond pattern. This method is applied to several ground-state structures of bacteriorhodopsin, which residues already present complicated titration behaviors in the absence of a proton gradient. Our calculations show that a pH gradient across the membrane influences in a non-trivial manner the protonation probabilities of six titratable residues which are known to participate in the proton transfer: D85, D96, D115, E194, E204, and the Schiff base. The residues connected to one side of the membrane are influenced by the pH on the other side because of their long-range electrostatic interactions within the protein. In particular, D115 senses the pH at the cytoplasmic side of the membrane and transmits this information to D85 and the Schiff base. We propose that the strong electrostatic interactions found between D85, D115, and the Schiff base as well as the interplay of their respective protonation states under the influence of a transmembrane pH gradient are responsible for the back-pressure effect on bacteriorhodopsin.     

Bacteriorhodopsin in liposomes. II. Experimental evidence in support of a theoretical model.

In the preceding article equations describing relevant ion flows in illuminated suspensions of bacteriorhodopsin liposomes have been derived. Here these equations are subjected to experimental tests. Changes in permeability characteristics of the liposomal membrane are brought about by addition of specific ionophores and change of medium composition. Using light-driven proton uptake and electrochemical potential differences for protons across the membrane as observation parameters, ridig attempts to falsify the derived equations are unsuccessful. Agreement between equations and experimental results is established on the point of: (i) the antagonistic effect of valinomycin and nigericin on the two components of the proton-motive force, (ii) the time dependence of the changes in transmembrane electrical and chemical potential differences after the onset of illumination. In three independent experimental systems evidence was obtained for the correctness of the postulated dependence of the turnover rate of the photochemical cycle on back pressure by the transmembrane electrochemical potential difference for protons.     

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.     

Energy-linked nicotinamide-nucleotide transhydrogenase. Light-driven transhydrogenase catalyzed by transhydrogenase from beef heart mitochondria reconstituted with bacteriorhodopsin

Purified nicotinamide-nucleotide transhydrogenase from beef heart mitochondria was co-reconstituted with bacteriorhodopsin to from transhydrogenase-bacteriorhodopsin vesicles that catalyze a 20-fold light-dependent and uncoupler-sensitive stimulation of the reduction of NADP+ and NADP+ analogs by NADH and a 50-fold shift of the nicotinamide nucleotide ratio. In the presence of light, the transhydrogenase-bacteriorhodopsin vesicles catalyzed a pronounced light intensity-dependent inward proton pumping as indicated by a pH shift of the medium. As indicated by pH shifts, proton pumping by the bacteriorhodopsin essentially paralleled the light-driven transhydrogenase. Addition of valinomycin increased the pH shift twice with a concomitant 50% inhibition of the light-driven transhydrogenase, whereas nigericin inhibited the pH shift completely and the light-driven transhydrogenase partially. Taken together, these results suggest that transhydrogenase and bacteriorhodopsin interact through a delocalized proton-motive force. Possible partial reactions of transhydrogenase were investigated with transhydrogenase-bacteriorhodopsin vesicles energized by light. Reduction of oxidized 3-acetylpyridine adenine dinucleotide by NADH, previously claimed to represent partial reactions, was found to require NADPH. Similarly, reduction of thio-NADP+ by NADPH required NADH. It is concluded that these reactions do not represent partial reactions.     

Binding of insulin to the external surface of liposomes: Effect of surface curvature,temperature, and lipid composition

The binding of insulin to the external surface of phosphatidylcholine liposomes as a function of the temperature, the surface curvature, and the composition of lipids was studied. The amount of the saturated binding of insulin to liposomes was assessed by gel-filtration chromatography. The binding of insulin to small unilamellar vesicles was highly dependent upon the temperature, favoring low temperatures. As the temperature increased, there was a distinct temperature range where the binding of insulin to small unilamellar vesicles decreased. The temperature ranges for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles were found to be 10–20°C and 21–37°C, respectively. These temperature ranges were quite different from the reported ranges of the gel → liquid crystalline phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) for DMPC or DPPC small unilamellar vesicles. In contrast to other proteins, the amount of insulin bound to DMPC and DPPC small unilamellar vesicles was negligible at or above the upper limit of the above temperature ranges, and increased steadily to 6–7 μmol of insulin per mmol of phospholipid as the temperature decreased to or below the lower limit of these temperature ranges. On the other hand, the binding of insulin to the large multilamellar liposomes cannot be detected at all temperatures tested. The affinity of insulin to neutral phosphatidylcholine small unilamellar vesicles appeared to be related to the surface curvature of the liposomes, favoring the liposomes with a high surface curvature. Furthermore, the amount of insulin bound to small unilamellar vesicles decreased as the content of the cholesterol increased. The presence of 10% molar fraction of phosphatidic acid did not appear to affect the binding of insulin to small unilamellar vesicles. However, the presence of 5% molar fraction of stearylamine in DPPC small unilamellar vesicles increased the amount of bound insulin as well as the extent of aggregation of liposomes. The results of the present study suggest that the interstitial regions of the acyl chains of phospholipids between the faceted planes of small unilamellar vesicles below $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ may be responsible for the hydrophobic interaction of insulin and small unilamellar vesicles. The tight binding of insulin to certain small unilamellar liposomes could lead to an overestimation of the true amount of insulin encapsulated in liposomes, if care is not taken to eliminate the bound insulin during the procedure of encapsulating insulin in liposomes.     

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.     

Formation of the transmembrane electrochemical potential on Staphylococcus cells and membrane vesicles

The generation of transmembrane difference of electrochemical potentials was registered on the intact cells and ultrasonication-obtained membrane vesicles of Staphylococcus aureus with the application of transmembrane electrophoresis of permeant anions, potassium transport in the presence of valinomycin and 8-anilinonaphthalene-1-sulphonate fluorescence. The membrane potential is formed when the chain of electron transfer or H+-ATPase functions or when the pH gradient varies (the nonenzymic pathway). M-chlorinecarbonylcyanidephenylhydrazonium, a protonophore uncoupler potassium cyanide, an inhibitor of the respiratory chain, N',N-dicyclohexylcarbodiimide, an inhibitor of ATPase, cause the membrane potential dissipation. The orientation of the transmembrane electric field is as follows: "minus" inside cells and "plus" inside membrane vesicles.     

Cation permeability induced by valinomycin,gramicidin D and amphotericin B in large lipidic unilamellar vesicles studied by 31P-NMR

Permeability induced by mobile carriers and channel-forming compounds in large unilamellar lipidic vesicles (LUV) has been studied by the proteon-cation exchange method. Proton movement has been monitored by pH-stat and ³¹P-NMR techniques. pH-stat measurements indicate that, in the presence of valinomycin, the proton efflux develops with a rate dependent upon valinomycin concentration, until equilibrium is reached. ³¹P-NMR spectra, monitoring pH-dependent intravesicular phosphate ionization, show that after addition of valinomycin the initial pH peak (pH 5.5; =0.25) shifts progressively to the position corresponding to the pH at equilibrium (pH 7.4; =2.20).In the presence of the channel-forming compounds, gramicidin D or amphotericin B, permeability developed in a few minutes whatever the concentration used. The percentage of total titratable proton released depends upon the antibiotic concentration. ³¹P-NMR spectra shows two signals from internal phosphate: one signal corresponding to the initial pH and a second signal corresponding to the pH at equilibrium indicating an all-or-none mode of action; just after addition of the antibiotic, two populations of vesicles coexist in proportions that depend on ionophore concentration; after longer incubation times all vesicles are permeabilized.The results obtained primarily reflect the differences in the mode of interaction with the membrane, of valinomycin as compared to the channel-forming reagents, gramicidin D or amphotericin B.     

Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves

The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K⁺. The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (ΔpH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K⁺-selective ionophore, valinomycin, stimulated uptake in the presence of a ΔpH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited ΔpH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H⁺/sucrose symporter in PM vesicles isolated from spinach leaves.