Accumulation of thallous ions (Tl+) as a measure of the electrical potential difference across the cytoplasmic membrane of bacteria.

The accumulation of thallous ions (204Tl+) by intact bacteria was investigated. I conclude that Tl+ is a permeant cation, and that it therefore accumulates in response to the electrical potential difference (delta psi) across the cytoplasmic membrane (interior negative). A comparison with other methods shows that the distribution ratio of 204Tl+ serves as a reasonably satisfactory method for measuring the membrane potential of Streptococcus faecalis. Glycolyzing cells of this organism develop membrane potentials of up to 180 mV. Preliminary experiments with Escherichia coli, especially those with a mutant defective in the proton-translocating ATPase, indicate that the Tl+ distribution also serves as a measure of the membrane potential in this organism. The particular advantage of Tl+ over other indicators of the membrane potential is that the cells need not be pretreated in any way. By use of the Tl+ distribution, it was calculated that respiring cells of E. coli develop a membrane potential of 160 mV with D-lactate and 180 mV with glucose as a substrate, respectively.     

Effect of energy metabolism on the ionic selectivity of the K+ center of the Na+/K+ pump in the skin of the frog

The uptake of Rb+ and Tl+ in frog skin incubated in saline containing 86Rb and 204Tl was studied. The ratio of ouabain-sensitive influxes Tl+/Rb+ = 2. Inhibition of the unidirectional transport of Na+ by rotenone or by Tl+ was not accompanied by the total decrease in the ouabain-sensitive uptake of Rb+ and Tl+. A substantial number of the Na+/K+ pumps continued to operate to maintain the intracellular ion homeostasis. The Tl+/Rb+ ratio of the cation influxes via pumps of this group was about twice as high compared to the control values. A coexistence of the two forms of Na+/K+ pumps in frog skin epithelium was postulated. They differ in both ion selectivity and the source of energy (respiration or glycolysis). The Tl+/Rb+ ratio of the ouabain-insensitive fluxes seems to be independent of the energetic metabolism.     

Uptake of thallous ions by mitochondria is stimulated by nonactin but not by respiration alone

Nonrespiring rat-liver mitochondria swell in media containing high concentrations of thallous nitrate, indicating passive penetration of Tl+. This swelling could be further stimulated by 10 nM or more nonactin while even 1 microM valinomycin was without effect. Nonactin was also much more potent than valinomycin in stimulating swelling of respiring mitochondria in the presence of thallous acetate. It is evident that nonactin acts as a potent ionophore of Tl+ able to promote both the passive and energized uptake of Tl+ in mitochondria. The distribution of Tl+, present in trace concentrations below 1 mM, was measured during energisation by respiration both in the presence and absence of ionophores. Respiration induced net uptake of Tl+ only in the presence of ionophores, though Tl+ as a permeant cation was expected to sense respiration-induced changes in the membrane potential. The data may be interpreted as indicating that no transmembrane potential is formed upon energisation, but localized fields, which are able to interact with the lipophilic ionophore complexes of Tl+, but not with the hydrophilic cation Tl+. This interpretation is valid only if thermodynamic equilibrium has been reached.     

The transport of univalent thallium across the human erythrocyte membrane

Transport of 204Tl was studied in human erythrocytes incubated in isotonic salt solutions at pH 7.4 and 37 degrees C. 204Tl was rapidly accumulated in cells up to the constant level within a 10 minutes incubation (t0.5 = 3.5 min). The rate of uptake and the distribution ratio decreased in the presence of 0.1 mM ouabain and 0.5-1.0 mM furosemide (t0.5 = 5 min). A broad variability of the coefficient 204Tl distribution was observed in the intact erythrocytes due to a ouabain-sensitive component which was seen to decrease with the increase in Tl+ concentration in the medium (0.005-0.2 mM), and also to depend on the medium ion composition. On the contrary, a passive distribution of 204Tl in the presence of ouabain and furosemide was relatively constant within 1.1-1.5. The steady state distribution of 204Tl was declined after a substitution of Cl- by sucrose in the medium due to depolarization of erythrocyte membrane. On the other side, 204Tl uptake by the cells was raised during hyperpolarization of the membrane in the presence of valinomycin.     

Measurement of plasma membrane and mitochondrial potentials in sea urchin sperm. Changes upon activation and induction of the acrosome reaction

The relationship between the plasma membrane potential and activation of sperm motility and respiration, or induction of the acrosome reaction, was explored in sperm of the sea urchin Strongylocentrotus purpuratus. Plasma and mitochondrial membrane potentials were estimated by measuring the uptake of [14C]thiocyanate ( [14C]SCN-) and [3H]tetraphenylphosphonium ( [3H]TPP+) in intact sperm and sperm made permeant with digitonin. Mitochondrial potentials up to-185 mV were found, consistent with data for TPP+ uptake into mitochondria from other cell types. Values for TPP+ uptake corrected for mitochondrial accumulation and estimates of SCN- uptake both indicated that the plasma membrane potential was about -30 mV for actively respiring sperm in seawater and about -60 mV for quiescent sperm in Na+-free seawater. Activation of sperm motility and respiration induced by Na+ increased the intracellular pH and caused a depolarization of both the plasma membrane and mitochondrial potentials. However, membrane potential depolarization did not occur when the activation was induced by increased extracellular pH or by the peptide speract, although activation was always linked to increased intracellular pH. The acrosome reaction, on the other hand, was always associated with sperm plasma membrane potential depolarization, whether it was induced by the physiological effector from the egg surface or by several artificial triggering regimens. Thus, activation of respiration and motility is primarily controlled by increased intracellular pH (Christen, R., Schackmann, R. W., and Shapiro, B. M. (1982) J. Biol. Chem. 257, 14881-14890), whereas the acrosome reaction also requires depolarization of the plasma membrane potential.     

Chemical modification of amino acid residues associated with the delta-4-3-ketosteroid-dependent photoinactivation of delta-5-3-ketosteroid isomerase.

We have studied the accumulation of dibenzyldimethyl-ammonium ion (DDA+) by respiring membrane vesicles of Escherichia coli, as an index of the generation of an electrical gradient during respiration. Nonrespiring vesicles accumulated DDA+ when K+ efflux was induced by valinomycin or monactin. By various criteria this was shown to be the exchange of one cation for another, independent of metabolism and coupled entirely by electrical forces. Uptake of DDA+ by respiring vesicles was inhibited by ionophores that translocate electrical charge and by reagents that block the respiratory chain. Oxamate and p-chloromercuribenzoate inhibited accumulation of DDA+ but did not dissipate a preformed pool; the reason appears to be that these reagents are less inhibitory to transport after lactate oxidation has begun than they are in resting vesicles. Uptake does not appear to involve a biological carrier, but requires trace amounts of a lipid-soluble anion such as tetraphenylboron, which has a catalytic role in DDA+ translocation. Respiring K+ vesicles accumulated substantially less DDA+ than did Na+ vesicles. Na+ was expelled from the vesicles concurrently with DDA+ uptake, whereas Rb+ and K+ were not. Thus, DDA+ uptake may be limited in the latter case by the availability of anionic groups. This explanation was supported by the finding that the addition of nigericin doubled the capacity of K+ vesicles to take up DDA+, presumably by providing a route for K+ to exit in exchange for H+. Parallel experiments on the valinomycin-dependent accumulation of Rb+ by respiring vesicles indicate that this process is analogous to the uptake of DDA+. Ionophores that elicit electrogenic K+ movement also induced respiration-linked transport. Proton-conducting ionophores and several inhibitors of respiration blocked Rb+ uptake and dissipated a preformed gradient. Preincubation of the vesicles with oxamate or p-chloromrecuribenzoate inhibited Rb+ uptake, but their addition to respiring vesicles again did not cause efflux. Rb+ and DDA+ be, but their addition to respiring vesicles again did not cause efflux. Rb+ and DDA+ compete for uptake when present simultaneously. We conclude that the accumulation of both DDA+ and Rb+ occurs in response to an electrical gradient, vesicle interior negative, produced by respiration.     

Thallium and rubidium permeability of human and rat erythrocyte membrane

Transport of Tl+ and Rb+ in human and rat erythrocytes was investigated in the presence of ouabain. The chloride-dependent cotransport of Tl+, Rb+ and Na+ was precluded by replacement of Cl- by NO3-. The inward and outward rate constants for the residual fluxes of the cations were determined by measuring the transport of 204Tl and 86Rb in double label experiments. The rate of passive transport of Tl+ exceeded that of Rb+ by one-two orders of magnitude in human as well as rat erythrocytes. The membrane barrier which contributes to the maintenance of ion gradients was shown not to be a barrier for Tl+ which easily penetrates the membrane by an unknown mechanism. In rat erythrocytes the barrier for Rb+ was 10-15 times weaker than that in human red blood cells, while the corresponding ratio of rat/human Tl+ permeabilities was about 1.8-2.0. It follows that Tl+ permeability is only slightly affected by factors modifying the permeability to alkali cations. The increase of temperature from 20 degrees to 37 degrees C resulted in a three-fourfold stimulation of the passive transport of Tl+ both in human and rat erythrocytes. The movement of Tl+ and Rb+ through the erythrocyte membrane differed substantially from their diffusion along the excitable membrane channels characterized both by poor Tl+/K+ selectivity and weak temperature dependence.     

Factors affecting the relative magnitudes of the ouabain-sensitive and the ouabain-insensitive fluxes of thallium ion in erythrocytes

A maximal rate of the ouabain-sensitive 204-Tl influx in human erythrocytes can be attained at trace concentrations of Tl+ in Mg2+ isotonic media free of K+ and Na+. The maximal influx of Tl+ from isotonic Mg(NO3)2 at 20 degrees C and pH 7.4 was 0.45 mM.l(-1).h-1 with a Km of 0.025 mM. In contrast to the active influx of Tl+, the passive Tl+ fluxes were neither saturated nor influenced by external cations in the range of concentrations of Tl+ and K+ studied. The rate constants of Tl+ passive fluxes in human and cat erythrocytes can be related to pH by the equation log kin(OUT)= -A + B.pH, where A and B are empirical constants for particular conditions. The apparent activation energy was 16 and 11 kcal/mol in sulphate and nitrate media, respectively. Tl+ and the alkali metal cations seem to overcome a common barrier in the erythrocyte membrane. Nevertheless, the rate of the passive penetration of Tl+ is about two orders of magnitude faster than those of K+ or Rb+. An extra non-Coulombic interaction between Tl+ and membrane ligands appears to be involved providing an accumulation of Tl+ somewhere in the vicinity of the membrane barrier and increasing the diffusion fluxes of Tl+ in both directions.     

The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential.

1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0' + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N'-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.     

Active transport of thallous ions by Streptococcus lactis.

Tl+ ions have been shown to mimic or compete with K+ in a number of membrane systems. We confirmed that in starved, valinomycin-treated cells of Streptococcus lactis 7962, Tl+ ions distributed themselves across the bacterial membrane in response to the potassium diffusion potential. In glucose-energized cells, however, Tl+ was taken up by a system specifically stimulated by sodium salts. The intracellular levels of Tl+ exceeded those attained by [3H]triphenylmethylphosphonium ion, a lipophilic cation which accumulates in response to the membrane potential. The uptake of Tl+ by (Na+ and glucose)-stimulated cells was strongly inhibited by potassium salts. These experiments suggest that metabolic energy is coupled to Tl+ transport by means of a high energy phosphate compound and that Tl+ ions are actively transported by a membrane carrier whose normal substrate is K+. The uptake of Tl+ is not a valid method for determining the streptococcal membrane potential.     

Effects of K+ on the proton motive force of Bradyrhizobium sp. strain 32H1.

In previous studies, respiring Bradyrhizobium sp. strain 32H1 cells grown under 0.2% O2, conditions that derepress N2 fixation, were found to have a low proton motive force of less than -121 mV, because of a low membrane potential (delta psi). In contrast, cells grown under 21% O2, which do not fix N2, had high proton motive force values of -175 mV or more, which are typical of respiring bacteria, because of high delta psi values. In the present study, we found that a delta psi of 0 mV in respiring cells requires growth in relatively high-[K+] media (8 mM), low O2 tension, and high internal [K+]. When low-[O2], high-[K+]-grown cells were partially depleted of K+, the delta psi was high. When cells were grown under 21% O2 or in media low in K+ (50 microM K+), the delta psi was again high. The transmembrane pH gradient was affected only slightly by varying the growth or assay conditions. In addition, low-[O2], high-[K+]-grown cells had a greater proton permeability than did high-[O2]-grown cells. To explain these findings, we postulate that cells grown under conditions that derepress N2 fixation contain an electrogenic K+/H+ antiporter that is responsible for the dissipation of the delta psi. The consequence of this alteration in K+ cycling is rerouting of proton circuits so that the putative antiporter becomes the major pathway for H+ influx, rather than the H+-ATP synthase.     

Respiratory-driven Na+ electrical potential in the bacterium Vitreoscilla

Vitreoscilla is a Gram-negative bacterium with unique respiratory physiology in which Na+ was implicated as a coupling cation for the generation of a transmembrane electrical gradient (delta psi). Thus, cells respiring in the presence of 110 mM Na+ generated a delta psi of -142 mV compared to only -42 and -56 mV for Li+ and choline, respectively, and even the -42 and -56 mV were insensitive to the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (DTHB). The kinetics of delta psi formation and collapse correlated well with the kinetics of Na+ fluxes but not with those of H+ fluxes. Cyanide inhibited respiration, Na+ extrusion, and delta psi formation 81% or more, indicating that delta psi formation and Na+ extrusion were coupled to respiration. Experiments were performed to distinguish among three possible transport systems for this coupling: (1) a Na(+)-transporting ATPase; (2) an electrogenic Na+/H+ antiport system; (3) a primary Na+ pump directly driven by the free energy of electron transport. DCCD and arsenate decreased cellular ATP up to 86% but had no effect on delta psi, evidence against a Na(+)-transporting ATPase. Low concentrations of DTHB had no effect on delta psi; high concentrations transiently collapsed delta psi, but led to a stimulation of Na+ extrusion, the opposite of that expected for a Na+/H+ antiport system. Potassium ion, which collapses delta psi, also stimulated Na+ extrusion. The experimental evidence is against Na+ extrusion by mechanisms 1 and 2 and supports the existence of a respiratory-driven primary Na+ pump for generating delta psi in Vitreoscilla.     

The relationship between extramitochondrial Ca2+ concentration, respiratory rate, and membrane potential in mitochondria from brown adipose tissue of the rat

The ability of isolated mitochondria from rat brown-adipose tissue to regulate extramitochondrial Ca2+ (measured by arsenazo) was studied in relation to their ability to produce heat (measured polarographically). The energetic state of the mitochondria was expressed as a membrane potential, delta psi (estimated with safranine), and was varied semi-physiologically by the use of different GDP concentrations. In these mitochondria GDP binds to the 32-kDa polypeptide, thermogenin, which regulates coupling. Ca2+ uptake (at 5 microM extramitochondrial Ca2+) was maximal at delta psi greater than 150 mV. Basal Ca2+ release increased from 1 to 2 nmol x min-1 x mg-1 below 150 mV. Na+ -stimulated rate of Ca2+ release was stable within the investigated delta psi span (100-160 mV). Initial Ca2+ levels were maintained below 0.2 microM for 100 mV less than delta psi less than 160 mV. Ca2+ levels maintained after Ca2+ challenge (20 nmol Ca2+ x mg-1) were below 0.4 microM for delta psi greater than 135 mM. Respiration was unstimulated for delta psi greater than 150 mV and was maximal at delta psi less than or equal to 135 mV. In the presence of well-oxidised substrates, the respiration at maximally activated thermogenin was markedly below fully uncoupled respiration and was probably limited by thermogenin activity--i.e. by a limited H+ reentry (OH- exit) and therefore by a membrane potential maintained at about 135 mV. It is concluded that at membrane potentials of 135 mV and above the mitochondria exhibit full Ca2+ control and are able to regulate thermogenic output up to maximum without interfering with this Ca2+ control. Membrane potential probably does not decrease below 135 mV in vivo. Therefore, Ca2+ homeostasis and thermogenesis are non-interfering and can be hormonally independently regulated, e.g. by alpha-adrenergic and beta-adrenergic stimuli, respectively.     

Hybrid respiration in the denitrifying mitochondria of Fusarium oxysporum

Induction of the mitochondrial nitrate-respiration (denitrification) system of the fungus Fusarium oxysporum requires the supply of low levels of oxygen (O(2)). Here we show that O(2) and nitrate (NO(3)(-)) respiration function simultaneously in the mitochondria of fungal cells incubated under hypoxic, denitrifying conditions in which both O(2) and NO(3)(-) act as the terminal electron acceptors. The NO(3)(-) and nitrite (NO(2)(-)) reductases involved in fungal denitrification share the mitochondrial respiratory chain with cytochrome oxidase. F. oxysporum cytochrome c(549) can serve as an electron donor for both NO(2)(-) reductase and cytochrome oxidase. We are the first to demonstrate hybrid respiration in respiring eukaryotic mitochondria.     

The effect of endogenous phosphate on the H+/Mn2+ ratio and the state of Mn2+ in the mitochondrial matrix.

1. Kinetics and stoichiometry of H+ extrusion and reuptake and of Mn2+ uptake and release have been measured in respiring liver mitochondria in the absence of external added Pi. H+ and Mn2+ fluxes are parallel during aerobic cation uptake but not during uncoupler induced cation release. The H+/Mn2+ is 1.24. Addition of SH reagents, in concentrations inhibiting the Pi carrier, modifies the kinetics of H+ extrusion and of Mn2+ uptake and release. The slow phase of uncoupler induced Mn2+ release is diminished. The H+/Mn2+ is increased to 1.72. Addition of SH reagents, after the phase of aerobic uptake is completed, results in a significant reduction of the extent of uncoupler-induced Mn2+ release. The extent of reuptake of endogenous Pi during aerobic uptake of Mn2+ is about 8 nmol x mg protein-1. 2. Aerobic uptake of Mn2+ in the absence of external Pi results in an electron spin resonance spectrum which is the sum of two components. One, denoted as S, corresponds to Mn(H2O)2+(6). Another denoted as E, reflects spin exchange narrowing. In contrast to previous claims the following evidence suggests that the spin exchange component is due to Mn3(PO4)2 precipitate: (a) the dimension of the spin exchange spectrum is markedly reduced by abolition of Pi transport; (b) the spin exchange spectrum is released very slowly by addition of uncouplers under conditions where uncouplers cause a rapid deenergization of mitochondria, reuptake of H+ and release of cations; (c) the free matrix Mn2+ is released slowly after addition of uncoupler if there is a large spin exchange signal; howeover the free matrix Mn2+ is abolished rapidly by uncoupler when formation of the spin exchange signal is prevented by pretreatment with Ca2+; (d) the band width of the spin exchange fraction is independent of the Mn2+/protein ratio either under kinetic or steady state conditions; (e) the experimental spectrum recalls closely that obtained by computer simulation by assuming it as a combination of Mn(H2O)2+(6) and Mn3(PO4)2. 3. It is concluded that endogenous Pi affects the process of aerobic divalent cation uptake. A part of Mn2+ uptake in the absence of externally added anions, consists of a Mn3(PO4)2 precipitate. This accounts for a H+/Mn2+ ratio lower than 2.     

Proton translocation coupled to formate oxidation in anaerobically grown fermenting Escherichia coli

Proton translocation, coupled to formate oxidation and hydrogen evolution, was studied in anaerobically grown fermenting Escherichia coli JW136 carrying hydrogenase 1 (hya) and hydrogenase 2 (hyb) double deletions. Rapid acidification of the medium by EDTA-treated anaerobic suspension of the whole cells or its alkalization by inverted membranes was observed in response to application of formate. The formate-dependent proton translocation and 2H(+)-K(+) exchange coupled to H(2) evolution were sensitive to the uncoupler, carbonylcyanide-m-chlorophenylhydrazone, and to copper ions, inhibitors of hydrogenases. No pH changes were observed in a suspension of formate-pulsed aerobically grown ("respiring") cells. The apparent H(+)/formate ratio of 1.3 was obtained in cells oxidizing formate. The 2H(+)-K(+) exchange of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes does take place in JW136 cell suspension. Hydrogen formation from formate by cell suspensions of E. coli JW136 resulted in the formation of a membrane potential (Deltapsi) across the cytoplasmic membrane of -130 mV (inside negative). This was abolished in the presence of copper ions, although they had little effect on the value of Deltapsi generated by E. coli under respiration. We conclude that the hydrogen production by hydrogenase 3 is coupled to formate-dependent proton pumping that regulates 2H(+)-K(+) exchange in fermenting bacteria.     

Hydrogen ion gradients across the mitochondrial, endosomal and plasma membranes in bloodstream forms of trypanosoma brucei solving the three-compartment problem.

Conditions for the use of both [14C]methylamine and 5, 5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin bloodstream forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99-7.03, the pH of the mitochondrial matrix was 7.71-7.73, and the algebraic average pH of the various endosomal compartments was 5.19-5.50. Similar values were found when cells were respiring aerobically on glycerol. However, bloodstream forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0-7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Deltapsip, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Deltapsip. Oligomycin abolished the mitochondrial pH gradient (DeltapHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (DeltapHe). The pH gradient across the plasma membrane (DeltapHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (DeltaPp) of -52 mV and an inwardly directed sodium-motive force (DeltaNp) of -149 mV, whereas the mitochondrial inner membrane had only an inwardly directed DeltaPm of -195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed DeltaPl within the range + 89 to +110 mV, depending on the measurement method.     

Effects of triorganotin-mediated anion-hydroxide exchange upon reconstituted cytochrome c oxidase proteoliposomes

Proteoliposomes containing cytochrome c oxidase and an internally trapped fluorescent pH probe (pyranine) were used to monitor respiration-dependent internal alkalinization and membrane potential formation. A maximum steady-state pH gradient of about 0.4 pH unit (vesicle interior alkaline) was obtained during active respiration in presence of reducing substrates and cytochrome c. This pH gradient was abolished by the triorganotin compounds tripropyl-, tributyl-, and triphenyl-tin chloride. At the same time, the membrane potential, measured by carbocyanine dye uptake, was slightly increased in value. Valinomycin, which abolishes the membrane potential, restores the value of delta pH at low trialkyltin concentrations. The organotin compounds acted as electroneutral ionophores which exchanged intravesicular OH- ions with external SCN-, I-, and CI- ions, but not NO3- or SO4(2-) ions. Abolition of delta pH is accompanied by an increase in respiration rate, but full respiratory stimulation only occurs when both delta psi and delta pH are abolished by addition of both triorganotin and valinomycin. The triorganotin-valinomycin combination leads to active KCl accumulation by the respiring proteoliposome, and it is necessary to postulate an electrically neutral KCl efflux process to explain the continued steady respiration of the proteoliposomes in the presence of this ionophore combination.     

Inhibition by dithioerythritol of alloxan induced efflux of Ca2+ and accompanying alterations in isolated liver mitochondria

Isolated mouse liver mitochondria respiring on succinate released Ca2+ when incubated with alloxan, accompanied by decreased membrane potential, stimulated state 4 respiration and swelling. All these effects of alloxan were inhibited by equimolar or higher concentrations of dithioerythritol (DTE), and in presence of added ATP a carboxyatractyloside-sensitive reuptake of Ca2+ was observed. The process of release and uptake of Ca2+ could be repeated by additional administrations of higher concentrations of alloxan and DTE plus ATP, respectively. The data suggest that the mitochondrial action of alloxan involves oxidation of membrane thiol groups.     

Effect of the membrane potential on the passive transport of Ca2+ in fractions of vesicles of the myometrium sarcolemma

Using a potential-sensitive fluorescent probe diS-C3-(5), the formation of the membrane (K+-diffusion) potential, delta psi, in the myometrium sarcolemmal vesicular fraction was demonstrated. The magnitude of this potential corresponds to that calculated according to the Nernst equation, is time-stable (characteristic dissociation time--3-5 min) and temperature-dependent and is generated upon the substitution of the anion (Cl- for gluconate-) and the compensating cation (Na+ for Tris+, choline+). The change in delta psi from -61 to 0 mV leads to the activation of passive Ca2+ efflux from the vesicles (with choline+ as the compensating cation in the dilution medium). At the same value of the potential, i. e., -61 mV, the substitution of choline in the dilution medium for Na+ or Li+ stimulates the passive release of Ca2+. Co2+, Mn2+ and D-600 suppress this process by 15-20% in depolarized vesicles which points to the inhibition of Ca2+ release with an alteration of the membrane potential value from 0 to -61 mV (20%). The potential-dependent component of passive Ca2+ transport is characterized by saturation with the substrate (Km = 0.5 mM). The dependence of Ca2+ flux release from the sarcolemmal vesicles on the membrane potential value (-60-+27 mV) is bell-shaped and qualitatively relative to the volt-amper characteristics of the steady state Ca2+ flux in single smooth muscle cells. Analysis of experimental results revealed that the potential-dependent component of passive Ca2+ transport in myometrium sarcolemmal vesicles is determined by the non-activated Ca2+ conductivity of plasma membrane.