Passive potassium ion permeability of Halobacterium halobium cell envelope membranes

Cell envelope vesicles, prepared from Halobacterium halobium, were loaded with 3 M KCl suspended in 3 M NaCl, and the loss of K⁺ was followed at various temperatures. The Arrhenius plot of the K⁺-efflux rates shows a break at 30°C, with higher energy of activation above the break. This temperature dependence is consistent with earlier studies of chain motions in liposomes prepared from isolated lipids. The efflux of K⁺ is more rapid with increasing pH between pH 5 and 7. Since these vesicles do not respire under the experimental conditions it was expected that the K⁺-efflux data would be related to the passive permeability of the membranes to K⁺. The apparent K⁺ permeability at 30°C is 1–2· 10^?10 cm·^?1. This value corresponds to a 5-h half-life for retained K⁺ in the envelope vesicles and to a probably much longer half-life in whole cells. The previously observed ability of Halobacterium to retain K⁺ in the absence of metabolism can thus be explained solely by the permeability characteristics of the membranes.     

Temperature dependence of action of polymyxin B on Escherichia coli

The temperature dependence of the action of polymyxin B on Escherichia coli was studied by using K+, Ca2+, and tetraphenylphosphonium (TPP+) ion-selective electrodes. At room temperature (27 degrees C), Ca2+ was released immediately after addition of polymyxin, while the efflux of K+ occurred after 30 s. The rapid release of Ca2+ was not affected by incubation temperature, while the efflux of K+ was significantly lowered at temperatures below about 25-30 degrees C. The uptake of TPP+ also increased after polymyxin addition. The release of Ca2+ and the uptake of TPP+ supported the disruption of the outer membrane structure reported previously. In experiments with isolated membrane vesicles (the cytoplasmic membrane being exposed), the efflux of K+ was not delayed, but was lowered at temperatures below about 15-20 degrees C. This temperature range differed significantly from that of whole cells, and was interpreted as representing a difference in membrane fluidity between the outer and cytoplasmic membranes. The phase transition temperature of the outer membrane is known to be higher than that of the cytoplasmic membrane; and the temperature dependence of efflux of K+ from membrane vesicles was compatible with the phase transition temperature of liposomes prepared with phospholipids (not containing lipopolysaccharides) extracted from E. coli. Thus, it was speculated that, with whole cells, polymyxin molecules passed through the outer membrane at temperatures above the phase transition and reached the cytoplasmic membrane, increasing its K+ permeability. The mechanism of the permeability change is discussed in terms of deformation of the cytoplasmic membrane structure induced by polymyxin molecules.     

Coupling between the bacteriorhodopsin photocycle and the protonmotive force in Halobacterium halobium cell envelope vesicles. III. Time-resolved increase in the transmembrane electric potential and modeling of the associated ion fluxes.

Bacteriorhodopsin functions as an electrogenic, light-driven proton pump in Halobacterium halobium. In cell envelope vesicles, its photocycle kinetics can be correlated with membrane potential. The initial decay rate of the M photocycle intermediate(s) decreases with increasing membrane potential, allowing the construction of a calibration curve. The laser (592.5 nm) was flashed at various time delays following the start of background illumination (592 +/- 25 nm) and transient absorbance changes at 418 nm monitored in cell envelope vesicles. The vesicles were loaded with and suspended in either 3 M NaCl or 3 M KCl buffered with 50 mM HEPES at pH 7.5 and the membrane permeability to protons modified by pretreatment with N,N'-dicyclohexylcarbodiimide. In each case the membrane potential rose with a halftime of approximately 75 ms. The steady-state potential achieved depends on the cation present and the proton permeability of the membrane, i.e., higher potentials are developed in dicyclohexylcarbodiimide treated vesicles or in NaCl media as compared with KCl media. The results are modeled using an irreversible thermodynamics formulation, which assumes a constant driving reaction affinity (Ach) and a variable reaction rate (Jr) for the proton-pumping cycle of bacteriorhodopsin. Additionally, the model includes a voltage-gated, electrogenic Na+/H+ antiporter that is active when vesicles are suspended in NaCl. Estimates for the linear phenomenological coefficients describing the overall proton-pumping cycle (Lr = 3.5 X 10(-11)/mol2/J X g X s), passive cation permeabilities (LHu = 2 X 10(-10), LKu = 2.2 X 10(-10), LNau = 1 X 10(-11)), and the Na+/H+ exchange via the antiporter (Lex = 5 X 10(-11)) have been obtained.     

Light-induced membrane potential and pH gradient in Halobacterium halobium envelope vesicles.

Illumination of envelope vesicles prepared from Halobacterium halobium cells causes translocation of protons from inside to outside, due to the light-induced cycling of bacteriorhodopsin. This process results in a pH gradient across the membranes, an electrical potential, and the movements of K+ and Na+. The electrical potential was estimated by following the fluorescence of a cyanine dye, 3,3'-dipentyloxadicarbocyanine. Illumination of H. halobium vesicles resulted in a rapid, reversible decrease of the dye fluorescence, by as much as 35%. This effect was not seen in nonvesicular patches of purple membrane. Observation of maximal fluorescence decreases upon ilumination of vesicles required an optimal dye/membrane protein ratio. The pH optimum for the lightinduced fluorescence decrease was 6.0. The decrease was linear with actinic light intensity up to about 4 X 10(5) ergs cn-2 s-1. Valinomycin, gramicidin, and triphenylmethylphosphonium ion all abolished the fluorescence changes. However, the light-induced pH change was enhanced by these agents. Conversely, buffered vesicles showed no pH change but gave the same or larger fluorescence changes. Thus, we have identified the fluorescence decrease with a light-induced membrane potential, inside negative. By using valinomycin-K+-induced membrane potentials, we calibrated the fluorescence decrease with calculated Nernst diffusion potentials. We found a linear dependence between potential and fluorescence decrease of 3 mV/%, up to 90 mV. When the envelope vesicles were illuminated, the total proton-motive force generated was dependent on the presence of Na+ and K+ and their concentration gradients across the membrane. In general, K+ appeared to be more permeable than Na+ and, thus, permitted development of greater pH gradients and lower electrical potentials. By calculating the total proton-motive force from the sum of the pH and potential terms, we found that the vesicles can produce proton-motive forces near--200 mV.     

Nigericin-mediated H+, K+ and Na+ transports across vesicular membrane: T-jump studies.

The decay of delta pH across vesicular membranes by nigericin-mediated H+ and metal ion (M+) transports has been studied at 25 degrees C after creating delta pH by temperature jump (T-jump). In these experiments K+ or Na+ were chosen as M+ for the compensating flux. Theoretical expressions derived to analyse these data suggest a method for estimating the intrinsic rate constants for the translocation of nig-H (k1) and for the translocation of nig-M (k2) across membrane, from the pH dependence of the delta pH decay. The following could be inferred from the analysis of data. (a) At pH approximately 7.5 and 250 mM ion concentrations, nigericin-mediated H+ and M+ transport rates are lower in a medium of K+ than in a medium of Na+, although ionophore selectivity of nigericin towards K+ is 25-45-times higher than that towards Na+. However, at lower [M+] (approximately 50 mM) the transport rates are higher in a medium of K+ than in a medium of Na+. Such behaviours can be understood with the help of parameters determined in this work. (b) The intrinsic rate constants k1 and k2 associated with the translocations of nig-H and nig-K or nig-Na across membrane are similar in magnitude. (c) At pH approximately 7.5 translocation of nig-H is the dominant rate-limiting step in a medium containing K+. In contrast with this, at this pH, translocation of nig-M is the dominant rate-limiting step when metal ion is Na+. (d)k1 approximately k2 approximately 6.10(3) s-1 could be estimated at 25 degrees C in vesicles prepared from soyabean phospholipid, and lipid mixtures of 80% phosphatidylcholine (PC) + 20% phosphatidylethanolamine and 92% PC + 8% phosphatidic acid. (e) The apparent dissociation constants of nig-M in vesicles were estimated to be approximately 1.5.10(-3) M for K+ and 6.4.10(-2) M for Na+ (at 50 mM ion concentrations) using approximately 10(-8.45) M for the apparent dissociation constant of nig-H.     

Light-induced glutamate transport in Halobacterium halobium envelope vesicles. I. Kinetics of the light-dependent and the sodium-gradient-dependent uptake.

During illumination Halobacterium halobium cell envelope vesicles accumulate [3H]glutamate by an apparently unidirectional transport system. The driving force for the active transport originates from the light-dependent translocation of protons by bacteriorhodopsin and is due to a transmembrane electrical potential rather than a pH difference. Transport of glutamate against high concentration gradients is also achieved in the dark, with high outside/inside Na+ gradients. Transport in both cases proceeds with similar kinetics and shows a requirement for Na+ on the outside and for K+ on the inside of the vesicles. The unidirectional nature of glutamate transport seems to be due to the low permeability of the membranes to the anionic glutamate, and to the differential cation requirement of the carrier on the two sides of the membrane for substrate translocation. Thus, glutamate gradients can be collapsed in the dark either by lowering the intravesicle pH (with nigericin, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone plus valinomycin), or by reversing the cation balance across the membranes, i.e., providing NaCl on the inside and KCl on the outside of the vesicles. In contrast to the case of light-dependent glutamate transport, the initial rates of Na+-gradient-dependent transport are not depressed when an opposing diffusion potential is introduced by adding the membrane-permeant cation, triphenylmethylphosphonium bromide. Therefore, it appears that, although the electrical potential must be the primary source of energy for the light-dependent transport, the translocation step itself is electrically neutral.     

Relationship between lipid fluidity and water permeability of bovine tracheal epithelial cell apical membranes

Apical membrane vesicles were prepared from bovine tracheal epithelial cells. These membranes were enriched in alkaline phosphatase specific activity 35-fold compared to cellular homogenates. Steady-state fluorescence polarization studies of these membranes, using three fluorophores, demonstrated that they possessed a relatively low fluidity. Studies using the probe 1,6-diphenyl-1,3,5-hexatriene detected thermotropic transitions at 25.7 +/- 0.4 and 26.8 +/- 0.6 degrees C in these membranes and their liposomes, respectively. Analysis of the composition of these membranes revealed a fatty acyl saturation index of 0.59 +/- 0.02, a protein/lipid ratio (w/w) of 0.60 +/- 0.06, a cholesterol/phospholipid ratio (mol/mol) of 0.83 +/- 0.11, and a sphingomyelin/lecithin ratio (mol/mol) of 0.64 +/- 0.10. Membrane vesicles were osmotically active when studied by a stopped-flow nephelometric technique. Arrhenius plots of rates of osmotic water efflux demonstrated break points at approximately 28 and 18 degrees C, with activation energies of 16.7 +/- 0.2 kcal mol-1 from 35 to 28 degrees C, 8.3 +/- 0.5 kcal mol-1 from 28 to 18 degrees C, and approximately 3.0 kcal mol-1 below 18 degrees C. Treatment of membrane vesicles with benzyl alcohol, a known fluidizer, decreased lipid order (increased fluidity) and increased the rate of osmotic water efflux. The present results suggest that water crosses tracheal epithelial cell apical membranes by solubility-diffusion across the lipid domain and that increases in fluidity correlate with increases in the water permeability of these membranes.     

The water and ionic permeability induced by polyene antibiotics across plasma membrane vesicles from Leishmania sp

An osmotic method has been used to study the effect of the polyene antibiotics amphotericin B, nystatin and candicidin on the water permeability of plasma membranes prepared from Leishmania sp. The effect of amphotericin B on the permeability of Leishmania membranes to a salt such as potassium nitrate was also investigated. A non-linear and saturable enhancement of water and salt permeability was measured with increasing polyene concentrations, which could be adjusted to Hill cooperativity equation. The antibiotic concentrations that induce at 30 degrees C half-maximal effects on the water permeability of Leishmania vesicles were 0.021 microM for candicidin, 0.21 microM for amphotericin B and 1.4 microM for nystatin. At 30 degrees C, the concentration of amphotericin B required to induce half of the maximal effect on the permeability of Leishmania vesicles to potassium nitrate was 1.8 microM. The temperature dependence for amphotericin B, nystatin and candicidin enhancement of the water permeability of Leishmania vesicles was determined by using Q10 data at 20 and 30 degrees C. The estimated activation energies at increasing polyene concentrations display the same general pattern for all three polyene antibiotics investigated, that is, a maximal positive value at about the polyene concentrations required for half-maximal effect. The significance of these results for understanding the mechanisms of action of polyene antibiotics on natural membranes is discussed.     

Salt tolerance of archaeal extremely halophilic lipid membranes

The membranes of extremely halophilic Archaea are characterized by the abundance of a diacidic phospholipid, archaetidylglycerol methylphosphate (PGP-Me), which accounts for 50-80 mol% of the polar lipids, and by the absence of phospholipids with choline, ethanolamine, inositol, and serine head groups. These membranes are stable in concentrated 3-5 m NaCl solutions, whereas membranes of non-halophilic Archaea, which do not contain PGP-Me, are unstable and leaky under such conditions. By x-ray diffraction and vesicle permeability measurements, we demonstrate that PGP-Me contributes in an essential way to membrane stability in hypersaline environments. Large unilamellar vesicles (LUV) prepared from the polar lipids of extreme halophiles, Halobacterium halobium and Halobacterium salinarum, retain entrapped carboxyfluorescein and resist aggregation in the whole range 0-4 m NaCl, similarly to LUV prepared from purified PGP-Me. By contrast, LUV made of polar lipid extracts from moderately halophilic and non-halophilic Archaea (Methanococcus jannaschii, Methanosarcina mazei, Methanobrevibacter smithii) are leaky and aggregate at high salt concentrations. However, adding PGP-Me to M. mazei lipids results in gradual enhancement of LUV stability, correlating with the PGP-Me content. The LUV data are substantiated by the x-ray results, which show that H. halobium and M. mazei lipids have dissimilar phase behavior and form different structures at high NaCl concentrations. H. halobium lipids maintain an expanded lamellar structure with spacing of 8.5-9 nm, which is stable up to at least 100 degrees C in 2 m NaCl and up to approximately 60 degrees C in 4 m NaCl. However, M. mazei lipids form non-lamellar structures, represented by the Pn3m cubic phase and the inverted hexagonal H(II) phase. From these data, the forces preventing membrane aggregation in halophilic Archaea appear to be steric repulsion, because of the large head group of PGP-Me, or possibly out-of-plane bilayer undulations, rather than electrostatic repulsion attributed to the doubly charged PGP-Me head group.     

Characterization of interaction between Tb3+ and porcine intestinal brush-border membranes

Effects of ionic strength and temperature on the interaction between Tb3+ and porcine intestinal brush-border membrane vesicles were studied. When Tb3+ was added to the vesicle suspension, Tb3+ fluorescence increased with increasing concentration of Tb3+, showing a saturation. The apparent dissociation constant of one of at least two components of this binding reaction was estimated to be about 12.5 microM at 25 degrees C, pH 7.4. But the affinity of Tb3+ for the membrane vesicles was variable with changes of ionic strength and temperature. The affinity was lowered by addition of KCl to medium and by increase of temperature above 30 degrees C. In addition, temperature-induced change in the affinity of Tb3+ for the membranes was reversible over a temperature range from 13 to 46 degrees C. Temperature-dependence profiles of the excimer formation efficiency of pyrene-labeled membranes and of the harmonic mean of the rotational relaxation times of pyrene molecules in the membranes revealed that the phase transition of the membrane lipids occurs at about 30 degrees C. Based on these results, characteristics of Tb3+ binding to the membranes are discussed in relation to the nature of lipid phase and surface charges of the membranes.     

Characteristics of (Na+ + K+)-ATPase in the gills of bass

A partial characterization of bass gill (Na+ + K+-ATPase is reported in the present paper. Microsomal preparation from gill homogenate showed optimal (Na+ + K+)-ATPase activity at pH 6,5 in the presence of 100 mM Na+, 20mM K+ and 5mM Mg2+. Under these conditions maximal activity was shown at 45 degrees C, even if an increased lability of the enzyme was shown at temperature greater than 30 degrees C. A complete inhibition of the enzyme occurred in the presence of 1 mM ouabain. The break in the Arrhenius plot occurred approximatively at the temperature of adaptation of these fish (18 degrees C). The energies of activation above and below the break were scarcely different from each other and lower than those reported in other Poikilotherms. Furthermore similar values of Km for Na+ were evidenced at 18 degrees C and 30 degrees C. The whole of data are discussed in comparison with other teleost gill (Na+ + K+)-ATPase reports and related to the physiological role of the enzyme in osmoregulation.     

Chloroplast Inner-Envelope ATPase Acts as a Primary H+ Pump

The stromal pH of the chloroplast must be maintained higher than that of the surrounding cytosol for photosynthetic carbon assimilation to occur. Experimental evidence demonstrating how this is accomplished in the plant cell is lacking. In the experiments reported here, we studied H+ and K+ flux across membranes of purified chloroplast inner-envelope vesicles. We were able to demonstrate ATP-dependent transport of both cations across the membranes of these vesicles. The data presented document the presence of an H+-pump ATPase in the chloroplast envelope. Energy-dependent K+ flux across these membranes occurs as a consequence of primary H+ pumping. The H+-pumping activity demonstrated in this report is consistent with a model involving the activity of this envelope ATPase as a primary mechanism facilitating a stroma:cytosol [delta]pH.     

Characterization of proton-transporting membranes from resting pig gastric mucosa

Membrane vesicles were purified from resting corpus mucosa of pig stomachs by velocity-sedimentation on a sucrose-Ficoll step gradient. Two vesicular fractions containing the (H+ + K+)-ATPase were obtained. One fraction was tight towards KCl, the other was leaky. At 21 degrees C maximal (H+ + K+)-ATPase activities of 0.8 and 0.4 mumol X mg-1 X min-1, respectively, were observed in lyophilized vesicles. The vesicles contained a membrane-associated carbonic anhydrase, the activity of which was in 100-fold excess of the maximal ATPase activity. Both vesicular fractions were rich in phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol. The characteristics of ion permeability and transport in the tight vesicles were in agreement with corresponding data for vesicles of a tubulovesicular origin in the parietal cell. Measurement of the rate of K+ uptake into the vesicles was based on the ability of K+ to promote H+ transport. The uptake was slow and dependent on the type of anion present. The effectiveness in promoting uptake of K+ by anions was SCN- greater than NO3- greater than Cl- much greater than HCO3- greater than SO4(2-). Uptake of K+ was much more rapid at alkaline pH than at neutral or at acidic pH. Addition of CO2 at alkaline pH strongly stimulated the rate of H+ accumulation in the vesicles. The initial part of this stimulation was sensitive to acetazolamide, an inhibitor of carbonic anhydrase. A model how the (H+ + K+)-ATPase and the carbonic anhydrase may co-operate is presented. It is concluded that membrane vesicles of a tubulovesicular origin can produce acid.     

Studies on K+ permeability of rat gastric microsomes

A population of gastric membrane vesicles of high K+ permeability and of lower density than endoplasmic tubulovesicles containing (H+-K+)-ATPase was detected in gastric mucosal microsomes from the rat fasted overnight. The K+-transport activity as measured with 86RbCl uptake had a Km for Rb+ of 0.58 +/- 0.11 mM and a Vmax of 13.7 +/- 1.9 nmol/min X mg of protein. The 86Rb uptake was reduced by 40% upon substituting Cl- with SO2-4 and inhibited noncompetitively by ATP and vanadate with a Ki of 3 and 30 microM, respectively; vanadate also inhibited rat gastric (H+-K+)-ATPase but with a Ki of 0.03 microM. Carbachol or histamine stimulation decreased the population of the K+-permeable light membrane vesicles, at the same time increased K+-transport activity in the heavy, presumably apical membranes of gastric parietal cells, and enabled the heavy microsomes to accumulate H+ ions in the presence of ATP and KCl without valinomycin. The secretagogue-induced shift of K+ permeability was blocked by cimetidine, a H2-receptor antagonist. Four characteristics of the K+ permeability as measured with 86RbCl were common in the resting light and the carbachol-stimulated heavy microsomes; (a) Km for +Rb, (b) anion sensitivity (Cl- greater than SO2-4), (c) potency of various divalent cations (Hg2+, Cu2+, Cd2+, and Zn2+) to inhibit Rb+ uptake, and (d) inhibitory effect of ATP, although the nucleotide sensitivity was latent in the stimulated heavy microsomes. The Vmax for 86RbCl uptake was about 10 times greater in the resting light than the stimulated heavy microsomes. These observations led us to propose that secretagogue stimulation induces the insertion of not only the tubulovesicles containing (H+-K+)-ATPase, but also the light membrane vesicles containing KCl transporter into the heavy apical membranes of gastric parietal cells.     

Permeability characteristics of erythrocyte ghosts prepared under isoionic conditions by a glycol-induced osmotic lysis.

A detailed study has been made of the permeability characteristics of human erythrocyte ghosts prepared under isoionic conditions by a glycol-induced lysis (Billah, M.M., Finean, J.B., Coleman, R. and Michell, R.H. (1976) Biochim. Biophys. Acta 433, 45-54). Impermeability to large molecules such as dextran (average molecular weight 70 000) was restored immediately and spontaneously after each of the 5-7 lyses that were required to remove all of the haemoglobin. Permeabilities to smaller molecules such as MgATP2-, [3H]inositol and [14C]choline were initially high but could be greatly reduced by incubation at 37 degrees C for an hour. The extent of such resealing decreased as the number of lyses to which the ghosts had been subjected increased. Both removal of haemoglobin and permeabilities to small molecules were affected significantly by pH, CA3+ concentrations and divalent cation chelators. Maximum resealing was achieved in ghosts prepared in the basic ionic medium (130 mM KCl, 10 nM NaCl, 2 mM MgCl2, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES)) at pH 7.0 (0 degrees C) and with a calcium level around 10(-5) M. Acidic pH facilitated the removal of haemoglobin whilst the presence of divalent cation chelators showed down its release. Retention of K+ by ghosts leaded with K+ during the first lysis and subsequently incubated at 37 degrees C was substantial but lation chelators slowed down its released. Retention of K+ by ghosts loaded with K+ during the first lysis and subsequently incubated at 37 degrees C was substantial but little K+ could be retained within the haemoglobin-free ghosts. Permeability of the ghosts to K+ after one lysis was affected by temperature, pH, Ca2+ concentrations and by the presence of divalent cation chelators.     

Structural and functional properties of a Ca2+-ATPase from human platelets

An antibody prepared against highly purified rabbit muscle Ca2+-ATPase from sarcoplasmic reticulum has been observed to cross-react with proteins in human platelet membrane vesicles. The antibody specifically precipitated Ca2+-ATPase activity from solubilized human platelet membranes and recognized two platelet polypeptides denatured in sodium dodecyl sulfate with Mr = 107,000 and 101,000. Ca2+-ATPase activity from Brij 78-solubilized platelet membranes was purified up to 10-fold. The purified preparation consisted mainly of two polypeptides with Mr approximately 100,000, and 40,000. The lower molecular weight protein appeared unrelated to Ca2+-ATPase activity. The Ca2+-ATPase in human platelet membrane vesicles exhibited "negative cooperativity" with respect to the kinetics of ATP hydrolysis. The apparent Km for Ca2+ activation of ATPase activity was 0.1 microM. Ca2+-dependent phosphorylation of platelet vesicles by [gamma-32P]ATP at 0 degrees C yielded a maximum of 0.2-0.4 nmol of PO4/mg of protein that was labile at pH 7.0 and 20 degrees C. This result suggests that only about 2-4% of the total protein in platelet membrane vesicles is the Ca2+-ATPase, which agrees with an estimate based on the specific activity of the Ca2+-ATPase in platelet membranes (20-50 nmol of ATP hydrolyzed/min/mg of protein at 30 degrees C). Calmodulin resulted in only a 1.6-fold stimulation of Ca2+-ATPase activity even after extensive washing of membranes with a calcium chelator or chlorpromazine. It is concluded that human platelets contain a Ca2+-ATPase immunochemically related to the Ca2+ pump from rabbit sarcoplasmic reticulum and that the enzymatic characteristics and molecular weight of the platelet ATPase are quite similar to those of the muscle ATPase.     

ATP synthesis in cell envelope vesicles of Halobacterium halobium driven by membrane potential and/or base-acid transition

Cell envelope vesicles active in ATP synthesis were prepared from Halobacterium halobium cells, which genetically lack bacteriorhodopsin, by sonication in the presence of substrates. ATP was synthesized when vesicles were illuminated to build up membrane potential through the action of halorhodopsin. The threshold value of membrane potential for ATP synthesis was about -100 mV relative to the external medium, i.e., inside-negative. ATP synthesis also occurred in the dark upon acidification of the external medium of a suspension of cell envelope vesicles. This base-acid transition ATP synthesis took place when the pH difference was greater than 1.6 units. The threshold pH difference was lowered when the base-acid transition was carried out under dim light which induced a membrane potential of about -100 mV. Regardless of the sort of driving force, ATP synthesis was optimum at the intravesicular pH of around 6.5 and almost nil at 8, where ATP syntheses by F0F1 type ATPases in other organisms are most active. The synthesis could be inhibited by N,N'-dicyclohexylcarbodiimide (DCCD) with a half-maximum inhibition at around 25 microM/2 mg protein/ml. These results strongly suggest that in halobacteria a DCCD-sensitive H+-translocating ATP synthase is in operation which is driven by membrane potential and/or pH gradient, and obeys chemiosmotic energetics. The results also suggest that the ATP synthase may not be identical to F0F1 type H+-translocating ATPases found in mitochondria, chloroplasts and eubacteria.     

Ion selectivity of temperature-induced and electric field induced pores in dipalmitoylphosphatidylcholine vesicles

Temperature and electric field are known to alter the permeability of the bilayer membrane in phospholipid vesicles. A study of cation selectivity of these membrane pores is reported for multilamellar liposomes (MLV) and unilamellar large vesicles (ULV, 95 +/- 5 nm diameter) of dipalmitoylphosphatidylcholine (DPPC). The permeability of ULV to Rb+ was 1.0 X 10(-6) micrograms/s at 22 degrees C and increased to 1.1 X 10(-5) micrograms/s at the gel to liquid-crystalline transition temperature (Tm) of the bilayer, at 42 degrees C. The permeability of ULV to Rb+ continued to increase beyond the Tm and reached 1.0 X 10(-4) micrograms/s at 56 degrees C, a 100-fold increase over the permeability at 22 degrees C. In contrast, the permeability of ULV to Na+ showed a local maximum of 6.0 X 10(-6) micrograms/s at 42 degrees C and decreased at temperatures higher or lower than the Tm. For MLV, the permeability to both Rb+ and Na+ peaked dramatically at the phase transition temperature, 42 degrees C, and subsided at lower and higher temperatures. When ULV were exposed to an electric field, the permeability to Rb+, Na+, and sucrose surged at a field strength of 30 kV/cm; 30 kV/cm can induce a transmembrane potential of 210 mV. In ULV, the electrically perforated lipid bilayer exhibited selectivity for Rb+ over Na+ only at a narrow electric field range, between 31 and 33 kV/cm. For MLV, no well-defined breakdown voltage was recorded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.     

Kinetic study of interaction between [14C]amphotericin B derivatives and human erythrocytes: relationship between binding and induced K+ leak

The relationship between polyene antibiotic binding to red cells and their membrane permeabilization was studied using two 14C-labelled amphotericin B (AmB) derivatives: N-fructosyl AmB and N-acetyl methyl ester AmB. The binding kinetics of both derivatives were determined on whole red cells and ghosts. The resulting experimental points were well fitted by monoexponential functions, and the characteristic t1/2 for both derivatives were calculated from these functions. At 2 X 10(-5) M, the half time t1/2 for N-acetyl methyl ester AmB (30.2 min) which forms aqueous aggregates was longer than the t1/2 for the more soluble species N-fructosyl AmB (4.5 min). At lower concentrations (10(-7) M), the t1/2 for N-acetyl methyl ester AmB (6.3 min) in a more solubilized form was close to that of N-fructosyl AmB (7.9 min). These results suggest that only solubilized species bound to red cell membranes and that disaggregation of aggregates is the limiting step in the binding process. The permeabilization of red cell membranes by N-fructosyl AmB, measured as intracellular K+ leak, was not instantaneous and at 10 degrees C external K+ was only detected 20 min after antibiotic addition. In contrast, binding occurs without lag time. Consequently, different mecanisms underlie binding and K+ permeability inducement. Absorption spectroscopy data showed that bound antibiotic is located in the hydrophobic membrane interior and that this penetration of the membrane by AmB derivatives occurs without lag time. Consequently, the lag time occurring for K+ permeability inducement would be due to some steps subsequent to binding and probably located in the hydrophobic membrane interior. This statement is further supported by the observation that the lag time is sensitive to changes in membrane fluidity as shown here by the break between 20 and 30 degrees C in the slope of the Arrhenius plot for the lag time, coinciding with the phase transition in red cell membranes.