pH equilibration in human erythrocyte suspensions

Summary A stopped-flow rapid reaction apparatus was used to study the rate of pH equilibration in human red cell suspensions. Flux of OH^– or H⁺ was determined over a wide range of extracellular pH (4–11) in CO₂-free erythrocyte suspensions. In these experiments, an erythrocyte suspension at pH 7.3 is rapidly mixed with an equal volume of NaCl solution at 3.0>pH>11.5. The pH of the extracellular fluid of the mixture changes rapidly as OH^– or H⁺ moves across the red cell membrane. Flux and velocity constants can be calculated from the initiald pH/dt using the known initial intra- and extracellular pH. It was found that the further the extracellular pH is from 7.3 (in either direction from 4–11), the greater the absolute value of total OH^– and/or H⁺ flux. Pretreatment with SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid), a potent anion exchange inhibitor, greatly reduces flux over the entire pH range, while exposure to valinomycin, a potassium ionophore, has no measurable effect. These data suggest that (i) both H⁺ and OH^– may be moving across the red cell membrane at all pH; (ii) the species dominating pH equilibration is probably dependent on the extracellular pH, which determines the magnitude of the driving gradient for each ion; and (iii) the rapid exchange pathway of the erythrocyte membrane may be utilized for both H⁺ and OH^– transport during CO₂-free pH equilibration.     

Arrangement of the electric potential-generating redox chain in the mitochondrial membrane

The intramembrane arrangement of the respiratory chain generating electric potential difference across the mitochondrial membrane has been studied. The accessibility of various respiratory carriers to the non-penetrating electron donors and acceptors, such as ferri-and ferrocyanide, cytochrome c. fumarate and nicotinamide nuclcotides has been used as a test for surface localization of the carrier in the membrane of mitochondria and inside-out (sonicated) submitochondrial particles. Membrane potential formation was detected by measuring the transmembrane flows of the penetrating anion, phenyl dicarbaundecaborne (PCB^–).It is shown that ferricyanide reduction can support PCB^– movement if this electron acceptor interacts with intact mitochondria in the region localized on the oxygen site of the antimycin-sensitive point. The same region is accessible for ferrocyanide whose oxidation by O₂ can be also coupled with PCB^– translocation. Added nicotinamide nuclcotides cannot be utilized by mitochondria for supporting PCB^– movement.PCB^– movement in the inside-out submitochondrial particles can be supported by reduction of ferricyanide or fumarate by NADH, and of NAD⁺ by NADPH, the former process being sensitive to rotenone but not to antimycin. Antimycin-insensitive reduction of feericyanide or of CoQ₆ by succinate is not coupled with PCB^– transport. Neither ferrocyanide nor ferrocytochromec can be used as electron donors in the particles.Penetrating electron donors (TMPDH₂, succinate) and acceptors (menadione) are effective both in mitochondria and particles.It is coucluded that flavin and transhydrogenase regions of the potential-generating redox chain are localized near the inner surface, cytochromec region-near the outers surface of the internal membrane of intact mitochondria. It means that the redox chain includes at least one act of the transmembrane transfer of reducing equivalents between flavins and cytochromec.     

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

Summary The uptake and binding of the lipophilic cations ethidium⁺, tetraphenylphosphonium⁺ (TPP⁺), triphenylmethylphosphonium⁺ (TPMP⁺), and tetraphenylarsonium⁺ (TPA⁺) in rat liver mitochondria and submitochondrial particles were investigated. The effects of membrane potential, surface potentials and cation concentration on the uptake and binding were elucidated. The accumulation of these cations by mitochondria is described by an uptake and binding to the matrix face of the inner membrane in addition to the binding to the cytosolic face of the inner membrane. The apparent partition coefficients between the external medium and the cytosolic surface of the inner membrane (K' _o) and the internal matrix volume and matrix face of the inner membrane (K' _i) were determined and were utilized to estimate the membrane potential from the cation accumulation factorR _c according to the relation =RT/ZF ln [(R _cV_o–K'_o)/(V_i+K'_i)] whereV _o andV _i are the volume of the external medium and the mitochondrial matrix, respectively, andR _c is the ratio of the cation content of the mitochondria and the medium. The values of estimated from this equation are in remarkably good agreement with those estimated from the distribution of⁸⁶Rb in the presence of valinomycin. The results are discussed in relation to studies in which the membrane potential in mitochondria and bacterial cells was estimated from the distribution of lipophilic cations.     

Inability of tributyltin-induced chloride/hydroxyl exchange to stimulate calcium transport in mitochondria isolated from flight muscle of the sheep blowfly Lucilia cuprina

Tributyltin in the concentration range 1–4μm failed to stimulate Ca²⁺ transport by Lucilia flight-muscle mitochondria in a medium containing KCl and respiratory substrate but devoid of P_i, despite its promotion of a rapid Cl⁻/OH⁻ exchange. When 2mm-P_i was present, concentrations of tributyltin greater than 1μm inhibited the initial rate of Ca²⁺ transport and induced efflux of the ion from the mitochondria in Cl⁻- or NO₃⁻-containing media. Lower concentrations had little effect. Oligomycin added at up to 10μg/mg of mitochondrial protein had no effect on Ca²⁺ transport. By contrast, approx. 0.3μm-tributyltin completely inhibited respiration supported by α-glycerophosphate in either the presence or absence of added ADP. The data suggest that tributyltin can inhibit Ca²⁺ transport in Lucilia flight-muscle mitochondria other than by facilitating a Cl⁻/OH⁻ exchange or producing an oligomycin-like effect.     

Electrically silent anion transport through bilayer lipid membrane induced by tributylin and triethyllead

Summary The method of the measurement of the nonelectrogenic fluxes of hydrogen (or hydroxyl) ions (J _H) based on the local proton gradients formation in the unstirred layers near a bilayer lipid membrane (BLM) is applied for recording the nonelectrogenic anion/OH^– exchange on BLM induced by tributyltin (TBT) and a novel carrier (Hager, A., Moser, I., & Berthold, W. 1987.Z. Naturforsch.,42C1116–1120), triethyllead (TEL). This method has been used previously for measuring the cation fluxes through BLM. TBT and TEL are shown to be equally efficient in the induction of Cl^–/OH^– exchange.J _H induced by TBT is constant at 4J _H decreases at pH<4 and pH>7. Both ionophores have a transport sequence: I^–> Br^–>Cl^–>F^–. The quatitative measurements reveal that TEL better discriminates these four anions than TBT. It is concluded that this method may prove helpful in a search and study of anion/OH^–-exchangers isolated from natural membranes.     

Anion specificity of the jejunal folate carrier: Effects of reduced folate analogues on folate uptake and efflux

Summary We previously reported that³H-folate uptake by rabbit jejunal brush-border membrane (BBM) vesicles was markedly stimulated by an outwardly directed OH^– gradient (pH_in 7.7, pH_out 5.5), inhibited by anion exchange inhibitors (DIDS, SITS, furosemide), and saturable (folateK _m=0.19 m) suggesting carrier-mediated folate/OH^– exchange (or H⁺/folate cotransport). In the present study, the anion specificity of this transport process was examined. Under conditions of an outwardly directed OH^– gradient, DIDS-sensitive folate uptake wascis inhibited (>90%) by reduced folate analogues: dihydrofolate (IC₅₀=0.40 m), folinic acid (IC₅₀=0.50 m), 5-methyltetrahydrofolate (IC₅₀=0.53 m), and (+)amethopterin (IC₅₀=0.93 M). In contrast, 10 m (–)amethopterin had only a modest effect on folate uptake (18% inhibition) suggesting stereospecificity of the folate/OH^– exchanger. The nonpteridine compounds which are transported by the folate carrier in L1210 leukemic cells (phthalate, thiamine pyrophosphate, and PO ₄ ^–3 ) did not inhibit jejunal folate uptake. Furthermore, folate uptake was not inhibited by SO ₄ ^–2 (4mm) or oxalate (4mm) thereby distinguishing this carrier from the previously described intestinal SO ₄ ^–2 /OH^– and oxalate/Cl^– exchangers. After BBM vesicles were loaded with³H-folate, the initial velocity of³H-folate efflux was stimulated by unlabeled folate in the efflux medium. The transstimulation of³H-folate efflux by unlabeled folate was furosemide (or DIDS) inhibitable and temperature sensitive. Half-maximal stimulation of furosemide-sensitive³H-folate efflux was observed with 0.25±0.05 m unlabeled folate, a concentration similar to theK _m for folate uptake. These data suggest that folate-stimulated³H-folate efflux is mediated by the folate/OH^– exchanger. With the exception of (–) amethopterin, reduced folate analogues also transstimulated furosemide-sensitive³H-folate efflux in a concentration-dependent manner suggesting stereospecific transport of these analogues by the folate/OH^– exchanger. In summary, folate transport by the jejunal folate/OH^– exchanger demonstrates bothcis inhibition and transstimulation by reduced folate analogues, but not by other inorganic or organic anions suggesting bidirectional transport of folate and a high degree of anion specificity.     

Lucigenin-derived chemiluminescence in intact isolated mitochondria

There are many data both in favor and against the use of lucigenin as a probe for superoxide anion (SA) in mitochondria, cells, and simple enzymatic systems. In the present work high concentrations (50-400 M) of lucigenin were used for continuous recording of rapid and reversible changes in the SA level in intact isolated mitochondria. The SA level in the presence of lucigenin rapidly and reversibly changed during the transition of the mitochondria from one functional state to another: under conditions of ATP synthesis from ADP and P_i, of Ca²⁺ accumulation, and of reverse electron transfer. Induction of a Ca²⁺,cyclosporin A-sensitive pore in mitochondria completely suppressed the lucigenin-derived chemiluminescence (LDC). The electron transfer in the Q-cycle of the respiratory chain complex III and high electric potential difference across the inner membrane of mitochondria were obligatory conditions for generation of a SA-dependent chemiluminescent signal. Based on our own and literature data, a scheme of LDC generation is suggested. The origin of superoxide anion detected in intact mitochondria with lucigenin is discussed.     

Active ion uptake and maintenance of cation-anion balance: A critical examination of their role in regulating rhizosphere pH

The processes responsible for maintenance of cation-anion balance in plants and their relation to active ion accumulation and changes in rhizosphere pH are outlined and discussed. The major processes involved are: (1) accumulation and degradation of organic acids which occur in the plant mainly as organic acid anions (and their transfer within the plant) and (2) extrusion of H⁺ or OH^– into the rhizosphere. The relative importance of the two processes is determined by the size of the excess anion or cation uptake. Indeed, plants typically absorb unequal quantities of nutritive cations (NH₄ ⁺+Ca²⁺+ Mg²⁺+K⁺+Na⁺) and anions (NO₃ ^–+Cl^–+SO₄ ^2–+H₂PO₄ ^–) and charge balance is maintained by excretion of an amount of H⁺ or OH^– which is stoichiometrically equal to the respective excess cation or anion uptake. The mechanisms and processes by which H⁺ and in particular OH^– ions are excreted in response to unequal cation-anion uptake are, however, poorly understood.The contemporary view is that primary active extrusion of H⁺, catalyzed by a membrane-located ATPase, is the major driving force for secondary transport of cations and anions across the plasma membrane. However, the fact that net OH^– extrusion often occurs (since excess anion absorption commonly takes place) implies there is a yet-to-be characterized OH^– ion efflux mechanism at the plasma membrane that is associated with anion uptake. There is, therefore, a need for future studies of the uptake mechanisms and stoichiometry of anion uptake; particularly that of NO₃ ^– which is often the predominant anion absorbed. Another related phenonenon which requires detailed study in terms of cation-anion balance is localized rhizosphere acidification which can occur in response to deficiencies of Fe and P.     

The mycotoxin ochratoxin A deranges pH homeostasis in Madin-Darby canine kidney cells

Ochratoxin A (OTA) is a nephrotoxin which blocks plasma membrane anion conductance in Madin-Darby canine kidney (MDCK) cells. Added to the culture medium, OTA transforms MDCK cells in a manner similar to exposure to alkaline stress. By means of video-imaging and microelectrode techniques, we investigated whether OTA (1 mol/liter) affects intracellular pH (pH.), Cl^– (Cl _i ^– ) or cell volume of MDCK cells acutely exposed to normal (pH_norm=7.4) and alkaline (pH_alk=7.7) conditions. At pH_norm, OTA increased Cl _i ^– by 2.6±0.4 mmol/liter (n=14, P<0.05) but had no effect on pH _i . At pH_alk, application of OTA increased Cl _i ^– by 8.6±2.6 mmol/liter (n=10, P< 0.05) and raised pH _i by 0.11±0.03 (n= 8, P<0.05). The Cl^–HCO ₃ ^– exchange inhibitor DNDS (4,4-dinitro-stilbene-2, 2-disulfonate; 10 mol/liter) eliminated the OTA-induced changes of pH _i and Cl _i ^– . OTA did not affect cell volume under both pH_norm and pH_alk conditions.We conclude that the OTA-induced blockade of plasma membrane anion conductance increases Cl _i ^– without changing cell volume. The driving force of plasma membrane Cl^–/HCO ₃ ^– exchange dissipates, leading to a rise of pH _i when cells are exposed to an acute alkaline load. Thus, OTA interferes with pH _i and Cl _i ^– homeostasis leading to morphological and functional alterations in MDCK cells.The work was supported by the Deutsche Forschungsgemeinschaft (DFG, Si 170/7-1).We thank the Zeiss Company (Oberkochen, Germany) for providing the Attofluor video-imaging system for the intracellular Ca²⁺ measurements.This study was carried out with the technical assistance of Sigrid Mildenberger and Ruth Freudinger.     

Complexes between uncouplers of oxidative phosphorylation

Summary We have examined the effects of two weak acid uncouplers of oxidative phosphorylation, 2,4-dinitrophenol and 5,6-dichloro-2-trifluoromethyl-benzimidazole, on the electrical properties of phospholipid bilayer membranes. All the effects they produce are consistent with the charged permeant species being a HA ₂ ^– complex formed between the neutral acid HA and its anion A^– and the current in the aqueous phases being carried by buffered hydrogen ions. When both uncouplers are present simultaneously, all the evidence we have obtained is consistent with the charged permeant species being a HAB^– complex formed between the neutral acid HA of one uncoupler and the anion B^– of the other. It was necessary, however, to take into account interfacial processes and the unstirred layers adjacent to the membrane, the adsorption of anions to the bilayer and the buffer level in the aqueous phases to explain the results in terms of this model. The degree to which these factors will complicate a comparison of results obtained on artificial systems and mitochondria is also discussed.     

Inhibition of anion transport across the mitochondrial membrane by amytal

It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K_i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p-trifluoromethoxyphenylhydrazone.Using [¹⁴C] succinate and [¹⁴C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P_i, succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P_i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [¹⁴C]succinate/anion (P_i, succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P_i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P_i translocation across the mitochondrial membrane.     

Influence of bovine serum albumine on the proton conductance of potato mitochondria membranes

Pulsed acid base titrations, according to the procedure of Mitchell and Moyle, have been carried out on potato mitochondria in the presence and absence of Bovine Serum Albumine (BSA). The rate of the pH decay is slower when BSA is present. The buffering capacities of the outer and inner phases, the t1/2 of the pH decay after an acid pulse and the proton conductance of the inner membrane have been measured. The results show that plant mitochondria are relatively impermeable to H⁺ and OH^–, but leakier than animal mitochondria. This may be related to the lower respiratory control ratios generally found with plant mitochondria.Abbreviations EGTA ethylene glycol bis (aminoethyl ether) NN tetraacetic acid - MERCAP sodium mercaptobenzothiazole - TRIS tris (hydroxymethyl) aminomethane - MOPS morpholinopropane sulfonic acid - BSA bovine serum albumine - RC respiratory control ratio     

Mechanism of mitochondrial transport of thallous ions

Rat liver mitochondria were found to swell under nonenergized conditions when suspended in media containing 30–40 mM TINO₃. Respiration on succinate caused a rapid contraction of mitochondria swollen under nonenergized conditions. In the presence of thallous acetate, there was a rapid initial swelling under nonenergized conditions until a plateau was reached; respiration on succinate then caused a further swelling. Trace amounts of²⁰⁴Tl (less than 100 µM) equilibrated fairly rapidly across the mitochondrial membrane. The influx of Tl⁺ was able to promote the decay not only of a valinomycin-induced K⁺-diffusion potential but also of respiration-generated fields in the inner membrane in accordance with the electrophoretic nature of Tl⁺ movement. Efflux of Tl⁺ showed a half-time of about 10 sec at 20°C and was not affected appreciably by the energy state. Efflux was retarded by Mg²⁺ and by lowering the temperature. The data indicate that Tl⁺ when present at high concentrations, 30 mM or more, distributes across the mitochondrial inner membrane both in response to electrical fields and to pH. In energized mitochondria the uptake of Tl⁺ would occur electrophoretically, while Tl⁺/H⁺ exchange would constitute a leak. In the presence of NO ₃ ^– , the movements of Tl⁺ are determined by that of NO ₃ ^– , indicating short-range coupling of electrical forces. At low concentrations of Tl⁺, 5 mM or less, there was no indication of a Tl⁺/H⁺ exchange, which appears to be induced by high concentrations of Tl⁺.     

Chloride distribution in the proximal convoluted tubule ofNecturus kidney

Summary To assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl^– activity ( _i ^Cl ) was studied in the proximal tubule ofNecturus kidney. Paired measurements of cell membrane PD (V _BL) and Cl-selective electrode PD (V _BL ^Cl ) were performed in single tubules, during reversible shifts of peritubular or luminal fluid composition. Steadystate _i ^Cl was estimated at 14.6±0.6 mmol/liter, a figure substantially higher than that predicted for passive distribution. To determine the site of the uphill Cl^– transport into the cell, an inhibitor of anion transport (SITS) was added to the perfusion fluid. Introduction of SITS in peritubular perfusate decreased _i ^Cl , whereas addition of the drug in luminal fluid slightly increased _i ^Cl ; both results are consistent with basolateral membrane uphill Cl^– transport from interstitium to the cell. TMA⁺ for Na⁺ substitutions in either luminal or peritubular perfusate had no effect on _i ^Cl . Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO ₃ ^– outflux), resulted in an increase of _i ^Cl , presumably related to enhanced Cl^– cell influx: we infer that Cl^– is exchanged against HCO ₃ ^– at the basolateral membrane. The following mechanism is suggested to account for the rise in luminal Cl^– concentration above equilibrium values: intracellular CO₂ hydration gives rise to cell HCO ₃ ^– concentrations above equilibrium. The passive exit of HCO ₃ ^– at the basolateral membrane energizes an uphill entry of Cl^– into the cell. The resulting increase of _i ^Cl , above equilibrium, generates downhill Cl^– diffusion from cell to lumen. As a result, luminal Cl^– concentration also increases.C.N.R.S. Greco 24. Part of this work was presented at the 12^th annual meeting of the American Society of Nephrology, Boston, Mass. (Edelman et al., 1979).     

Dual Effect of Phosphate Transport on Mitochondrial Ca2+ Dynamics

The large inner membrane electrochemical driving force and restricted volume of the matrix confer unique constraints on mitochondrial ion transport. Cation uptake along with anion and water movement induces swelling if not compensated by other processes. For mitochondrial Ca²⁺ uptake, these include activation of countertransporters (Na⁺/Ca²⁺ exchanger and Na⁺/H⁺ exchanger) coupled to the proton gradient, ultimately maintained by the proton pumps of the respiratory chain, and Ca²⁺ binding to matrix buffers. Inorganic phosphate (P_i) is known to affect both the Ca²⁺ uptake rate and the buffering reaction, but the role of anion transport in determining mitochondrial Ca²⁺ dynamics is poorly understood. Here we simultaneously monitor extra- and intra-mitochondrial Ca²⁺ and mitochondrial membrane potential (ΔΨ_m) to examine the effects of anion transport on mitochondrial Ca²⁺ flux and buffering in P_i-depleted guinea pig cardiac mitochondria. Mitochondrial Ca²⁺ uptake proceeded slowly in the absence of P_i but matrix free Ca²⁺ ([Ca²⁺]_mito) still rose to ∼50 μm. P_i (0.001–1 mm) accelerated Ca²⁺ uptake but decreased [Ca²⁺]_mito by almost 50% while restoring ΔΨ_m. P_i-dependent effects on Ca²⁺ were blocked by inhibiting the phosphate carrier. Mitochondrial Ca²⁺ uptake rate was also increased by vanadate (V_i), acetate, ATP, or a non-hydrolyzable ATP analog (AMP-PNP), with differential effects on matrix Ca²⁺ buffering and ΔΨ_m recovery. Interestingly, ATP or AMP-PNP prevented the effects of P_i on Ca²⁺ uptake. The results show that anion transport imposes an upper limit on mitochondrial Ca²⁺ uptake and modifies the [Ca²⁺]_mito response in a complex manner.     

Role of substrate binding forces in exchange-only transport systems: II. Implications for the mechanism of the anion exchanger of red cells

Summary The transition-state theory of exchange-only membrane transport is applied to experimental results in the literature on the anion exchanger of red cells. Two central features of the system are in accord with the theory: (i) forming the transition state in translocation involves a carrier conformational change; (ii) substrate specificity is expressed in transport rates rather than affinities. The expression of specificity is consistent with other evidence for a conformational intermediate (not the transition state) formed in the translocation of all substrates. The theory, in conjunction with concepts derived from the chemistry of macrocyclic ion inclusion complexes, prescribes certain essential properties in the transport site. Separate substites are required for the preferred substrates. Cl^– and HCO ₃ ^– , to account for tight binding in the transition state (K _diss1m). Further, the following mechanism is suggested. A substrate anion initially forms a loose surface complex at one subsite, but in the transition state the subsites converge to form an inclusion complex in which the binding forces are greatly increased through a chelation effect. The conformational change at the substrate site, which is driven by the mounting forces of binding, sets in train a wider conformational change that converts the carrier from an immobile to a mobile form. Though simple, this composite-site mechanism explains many unsual features of the system. It accounts for substrate inhibition, partially noncompetitive inhibition of one substrate by another, and tunneling, which is net transport under conditions where exchange should prevail, according to other models. All three types of behavior result from the formation of a ternary complex in which substrate anions are bound at both subsites. The mechanism also accounts for the enormous range of substrate structures accepted by the system, for the complex inhibition by the organic sulfate NAP-taurine, and for the involvement of several cationic side chains and two different protein domains in the transport site.     

Inner voltage clamping. A method for studying interactions among hydrophobic ions in a lipid bilayer

Ketterer, et al. (1971) have suggested that a combination of electrostatic and chemical interactions may cause hydrophobic ions absorbed within a bilayer lipid membrane to reside in two potential wells, each close to a membrane surface. The resulting two planes of charges would define three regions of membrane dielectric: two identical outer regions each between a plane of absorbed charges and the plane of closest approach of ions in the aqueous phase; and the inner region between the two planes of adsorbed charges. The theory describing charge translocation across the inner region is based on a simple three-capacitor model. A significant theoretical conclusion is that the difference between the voltage across the inner region, V_i, and the voltage across the entire membrane, V_m, is directly proportional to the amount of charge that has flowed in a voltage clamp experiment. We demonstrate that we can construct an “inner voltage clamp” that can maintain, with positive feedback, a constant inner voltage, V_i. The manifestation of proper feedback is that the clamp current (after a voltage step) will exhibit pure (i.e., single time-constant) exponential decay, because the voltage dependent rate constants governing translocation will be independent of time. The “pureness” of the exponential is maximized when the standard deviation of the least-square fit of the appropriate exponential equation to the experimental data is minimized. The concomitant feedback is directly related to the capacitances of the inner and outer membrane regions, C_i and C_o.

Experimental results with tetraphenylborate ion adsorbed in bacterial phosphatidylethanolamine/n-decane bilayers indicate C_i ~ 5 · 10^-7F/cm² and C_o ≈ 5 · 10^-5F/cm².

     

Proton conductance through phospholipid bilayers: Water wires or weak acids?

The proton/hydroxide (H⁺/OH^–) permeability of phospholipid bilayer membranes at neutral pH is at least five orders of magnitude higher than the alkali or halide ion permeability, but the mechanism(s) of H⁺/OH^– transport are unknown. This review describes the characteristics of H⁺/OH^– permeability and conductance through several types of planar phospholipid bilayer membranes. At pH7, the H⁺/OH^– conductances (G _H/OH) range from 2–6 nS cm^–2, corresponding to net H⁺/OH^– permeabilities of (0.4–1.7)×10^–5 cm sec^–1. Inhibitors ofG _H/OH include serum albumin, phloretin, glycerol, and low pH. Enhancers ofG _H/OH include chlorodecane, fatty acids, gramicidin, and voltages >80 mV. Water permeability andG _H/OH are not correlated. The characteristics ofG _H/OH in fatty acid (weak acid) containing membranes are qualitatively similar to the controls in at least eight different respects. The characteristics ofG _H/OH in gramicidin (water wire) containing membranes are qualitatively different from the controls in at least four different respects. Thus, the simplest explanation for the data is thatG _H/OH in unmodified bilayers is due primarily to weakly acidic contaminants which act as proton carriers at physiological pH. However, at low pH or in the presence of inhibitors, a residualG _H/OH remains which may be due to water wires, hydrated defects, or other mechanisms.     

A channel that allows inwardly directed fluxes of anions in protoplasts derived from wheat roots

An anion channel that only allows outward current flow (anion influx) has been identified in protoplasts derived from wheat (Triticum aestivum L., Triticum turgidum L.) roots. The anion outward rectifier (anion OR) measured by patch-clamp of whole cells activated very quickly, usually reaching a steady-state level in less than 100 ms and was easily distinguished from the cation outward rectifier (cation OR) which activated more slowly during membrane depolarisation. The anion OR is permeable to NO ₃ ^– and Cl^–, moderately permeable to I^–, and relatively impermeable to H₂PO₄/^– and ClO₄/^–. An anomalous mole-fraction effect between ClO_4/ ^– and Cl^– was observed on the outward current, indicating that the channel is a multi-ion pore. The anion OR is gated by both voltage and external anion concentration such that it activates near to the equilibrium potential for the permeant anion. It activated at more negative membrane potentials when NO ₃ ^– was substituted for Cl^– in the external medium, indicating that the channel may function to allow NO ₃ ^– influx under luxuriant external NO ₃ ^– concentrations. For most experiments, K⁺ and Cl^– were the main cation and anion in solution, and under these conditions it appeared likely that the anion OR functioned in membrane-potential regulation by facilitating a Cl^– influx at membrane potentials more positive than the chloride reversal potential (E_Cl). If E_Cl was more negative than the K⁺ reversal potential (E_K) then the anion OR dominated but both the anion and cation ORs occurred together when the membrane potential difference (V_m) was positive of both E_Cl and E_K. The cation OR was inhibited by increasing external Cl^– concentrations, but the anion OR was not affected by external K⁺ or Na⁺ concentration. The anion-transport inhibitors, zinc and phenylglyoxal were ineffective in blocking the anion OR. 4,4-Di-isothiocyanostilbene-2, 2-disulfonic acid (DIDS) irreversibly blocked about 34% of the current when applied extracellularly at a concentration of 25 M, and about 69% at a concentration of 200 M. However, DIDS (200 M) also occasionally acted as an irreversible blocker of the cation OR. Perchlorate blocked irreversibly 75% of the current at an external concentration of 10 mM and did not block the cation OR. Whole-cell currents also indicated that the anion OR was insensitive to external pH (pH=5–7) and calcium concentration ([Ca²⁺]=0.1–10 mM). Increasing intracellular calcium concentration significantly increased the occurrence of the fast outward current in whole cells (P < 0.005, X² test). With approximately 10 nM calcium inside the cell the anion outward current was observed in 64% (n = 45) of cells and with 50 nM calcium inside the cell the anion current was observed in 88% (n = 69) of cells. Single-anion OR channels observed in outside-out patches had a conductance in 300 mM KCl (external) of about 4 pS. When voltage pulses were applied to outside-out patches the average currents were similar to those observed in whole cells. The significance of the anion OR as a likely route for Cl uptake in high salinities is discussed.Abbreviations Bath solution bathing the extracellular face of the membrane - DIDS (4,4-diisothiocyanostilbene-2,2-disulfonic acid) - E_x reversal potential for ion x - OR outward rectifier - Pip solution inside the pipette - TEACl (tetraethyl-ammonium chloride) - V_m membrane potential difference We thank the Australian Research Council for financial support, G.P. Findlay and A. Garrill for helpful discussions, and K. Morris and D. Mackenzie for expert technical assistance. M.S. was supported by an Australian Postgraduate Research Award.     

Optimal temperature and concentration profiles in a cascade of CSTR's performing Michaelis-Menten reactions with first order enzyme deactivation

A necessary condition is found for the intermediate temperatures and substrate concentrations in a series of CSTR's performing an enzyme-catalyzed reaction which leads to the minimum overall volume of the cascade for given initial and final temperatures and substrate concentrations. The reaction is assumed to occur in a single phase under steady state conditions. The common case of Michaelis-Menten kinetics coupled with first order deactivation of the enzyme is considered. This analysis shows that intermediate stream temperatures play as important a role as intermediate substrate concentrations when optimizing in the presence of nonisothermal conditions. The general procedure is applied to a practical example involving a series of two reactors with reasonable values for the relevant five operating parameters. These parameters are defined as dimensionless ratios involving activation energies (or enthalpy changes of reaction), preexponential factors, and initial temperature and substrate concentration. For negligible rate of deactivation, the qptimality condition corresponds to having the ratio of any two consecutive concentrations as a single-parameter increasing function of the previous ratio of consecutive concentrations.List of Symbols C _E,0 mol.m^–3 Initial concentration of active enzyme - C _E,i mol.m^–3 Concentration of active enzyme at the outlet of the i-th reactor - C _S,0 mol.m^–3 Initial concentration of substrate - C _S,i mol.m^–3 Concentration of substrate at the outlet of the i-th reactor - Da _i Damköhler number associated with the i-th reactor ((V _i.k_v,0.C_E,0)/(Q.C_S,0)) - Da _min Minimum value of the overall Damköhler number - Da _tot Overall Damköhler number - E _d J.mol^–1 Activation energy of the step of deactivation of the enzyme - E _m J.mol^–1 Standard enthalpy change of the step of binding of substrate to the enzyme - E _v J.mol^–1 Activation energy of the step of enzymatic transformation of substrate - i Integer variable - j Dummy integer variable - k Dummy integer variable - k _d,i s^–1 Kinetic constant associated with the deactivation of enzyme in the i-th reactor (k _d,o·exp{–E _d/(R.T _i}) - k _d,0 s^–1 Preexponential factor of the kinetic constant associated with the deactivation of the enzyme - K _m,i mol.m^–3 Equilibrium constant associated with the binding of substrate to the enzyme in the i-th reactor, (k _m,o·exp{–E _m}(R.T _i}) - K _m,0 mol.m^–3 Preexponential factor of the Michaelis-Menten constant associated with the binding of substrate to the enzyme - k _v,i s^–1 Kinetic constant associated with the transformation of the substrate by the enzyme in the i-th reactor (k _v,o·exp{–E _v/(R.T _i})) - k _v,0 s^–1 Preexponential factor of the kinetic constant associated with the transformation of the substrate by the enzyme - N Number of reactors in the series - Q m³.s^–1 Volumetric flow rate of reacting liquid through the reactor network - R J.K^–1.mol^–1 Ideal gas constant - T _i K Absolute temperature at the outlet of the i-th reactor - T ₀ K Initial absolute temperature - V _i m³ Volume of the i-th reactor - v _max mol.m^–3.s^–1 Maximum rate of reaction under saturation conditions of substrate - x _i Normalized concentration of substrate (C_S,i/C_{S, 0}) - x _i,opt Optimum value of the normalized concentration of substrate - y _i Dimensionless temperature (exp{–T ₀/T _i}) - y _i,opt Optimum value of the dimensionless temperature Greek Symbols Dimensionless preexponential factor associated with the Michaelis-Menten constant (K _m,0/C_s,0) - Dimensionless activation energy of the step of enzymatic transformation of substrate (E _v/R.T₀)) - Dimensionless standard enthalpy change of the step of binding of substrate to the enzyme (E _m/(R.T₀)) - Dimensionless activation energy of the step of deactivation of the enzyme (E _d/(R.T₀)) - Dimensionless deactivation preexponential factor ((k _d,0.C_S,0)/(k_v,0.C_E,0)