Showing papers on "Electrochemical gradient published in 1979"

Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state.

Abstract: The membrane potential of mitochondria was estimated from the accumulation of tetraphenyl phosphonium (TPP+), which was determined with the TPP+-selective electrode developed in the present study. The preparation and some operational parameters of the electrode were described. The kinetics for uptake by mitochondria of TPP+ and DDA+ (dibenzyldimethyl ammonium) were analyzed, and it was found that TPP+ permeated the mitochondrial membrane about 15 times faster than DDA+. The final amounts of accumulation of TPP+ and DDA+ by mitochondria were approximately equal. For the state-4 mitochondria, the membrane potential was about 180 mV (interior negative). Simultaneous measurements of TPP+-uptake and oxygen consumption showed that the transition between states 3 and 4 was detectable by use of the TPP+-electrode. After the TPP+-electrode showed that state-4 was reached, the extra-mitochondrial phosphorylation potential was measured. The difference in pH across the membrane was measured from the distribution of permeant anion, acetate, so as to calculate the proton electrochemical potential. The ratio of extra-mitochondrial phosphorylation potential to proton electro-chemical potential, n was close to 3. This value of n was also found to be 3 when ATP was hydrolyzed under the condition that the respiratory chain was arrested. The implication that n = 3 was discussed.

The measurement of membrane potential and deltapH in cells, organelles, and vesicles.

Abstract: Publisher Summary The determination of both membrane potential and pH gradient (ΔpH) across a membrane allows the calculation of the proton electrochemical potential difference, or the proton-motive force. The value of the proton-motive force is important for an experimental evaluation of the chemiosmotic hypothesis. It is also an important indicator of the coupling in energy-conversion membrane systems. It is the most sensitive and quantitative indicator of coupling in these systems. As the proton-motive force can be generated by various means, the degree of coupling can be evaluated in systems that lack the complete machinery for oxidative phosphorylation or photophosphorylation. This is in contrast to the more conventional parameters of coupling, such as phosphate/oxygen (P/O), respiratory control, and phosphate potential. The determination of membrane potential or internal pH is not limited to systems of energy conversion; the chapter describes methods for determining the membrane potential in suspensions of cells, organelles, or vesicles.

Energy interconversion by the Ca2+-dependent ATPase of the sarcoplasmic reticulum.

Abstract: PERSPECTIVES AND SUMMARY 276 INTERCONVERSION OF CaH BINDING SITES OF DIFFERENT AFFINITIES 277 A BRIEF ACCOUNT OF ATP HYDROLYSIS AND CaH TRANSPORT 278 THE REVERSAL OF THE CaH PUMP 280 ATP Synthesis 281 ATP � Pi Exchange 282 Pi � HOH EXCHANGE AND PHOSPHORYLATION OF THE CaH-DEPENDENT ATPase BY Pi IN THE ABSENCE OF CaH CONCENTRATION GRADIENT 282 SIMULTANEOUS PHOSPHORYLATION OF THE CaH-DEPENDENT ATPase BY NTP AND Pi 284 PHOSPHATE TRANSFER FROM THE PHOSPHO ENZYME TO ADP 285 Pi DEPENDENCE 286 NET SYNTHESIS OF ATP IN THE ABSENCE OF A CaH GRADIENT 287 RATE OF PHOSPHOENZYME HYDROLYSIS 288 PROTON GRADIENT AND SYNTHESIS OF ATP BY THE Ca2+DEPENDENT ATPase 289

Physiological role of ATP-driven calcium pump in squid axon

Abstract: IT has been proposed that the regulation of the ionised calcium concentration inside many excitable and non-excitable cells is mediated by a Na–Ca countertransport mechanism where the Na electrochemical gradient provides the energy to maintain low levels of intracellular Ca2+ (refs 1, 2). Two recent observations, however, provide evidence consistent with the idea that Ca extrusion in squid axons is mediated, at least in part, by an ATP-driven Ca pump of the type observed in red blood cells3,4. First, in the absence of a Na gradient across the membrane there is an ATP-dependent ‘uphill’ extrusion of Ca which persists in the absence of external Na, Ca and Mg (ref. 3). And second, in Ca-injected unpoisoned axons, 50–90% of the Ca efflux can occur as an ‘uncoupled’ flux—that is, Ca efflux not accompanied by the uptake of Na, Ca or Mg (ref. 5). The existence of two apparently separate mechanisms for Ca transport, Na–Ca exchange and an ATP driven Ca-pump, raises the question of whether, in normal conditions, both mechanisms participate in the maintenance of the physiological pCa. To be of any use in controlling the pCa of the nerve, the mechanism responsible must be able to operate at normal Ca2+ concentrations, <10−7 M (ref. 6). We now present evidence showing (1) that in squid axons with physiological levels of Ca2+i most of the Ca efflux can be accounted for by an ATP-dependent system with high affinity for Ca2+i and ATP and which can operate in the complete absence of external Na and Ca; and (2) that a Ca transport mechanism with a low affinity for Ca2+i and ATP and extremely sensitive to Nai, Nao and Cao can contribute substantially to the total Ca efflux only at Ca2+i well above the physiological Ca2+ concentrations.

Direct addition of insulin inhibits a high affinity Ca2+-ATPase in isolated adipocyte plasma membranes.

Abstract: The mechanism by which insulin regulates cellular metabolism remains unknown although indirect evidence suggests that alterations in intracellular calcium are important. More specifically, it has been proposed that insulin triggers an increase in intracellular calcium which is responsible for the subsequent modification of metabolic activities1,2. The cell maintains a large electrochemical gradient for ionised calcium between the cytoplasm ( 10−3 M). The plasma membrane may, therefore, be important in the regulation of calcium homeostasis, as a slight alteration in the processes maintaining this gradient could result in marked changes in cytoplasmic calcium. One such process is the active extrusion of calcium from the cell by a high affinity calcium-stimulated ATPase (Ca2+-ATPase). Such a mechanism has been well established in red cells5 and is postulated in nerve6, liver7 and muscle8. We have identified a high affinity Ca2+- ATPase in a plasma membrane-enriched subcellular fraction isolated from rat adipocytes9 which may provide the enzymatic basis for a calcium extrusion pump. We demonstrate here that the Ca2+-ATPase is specifically inhibited by the direct addition of physiological concentrations of insulin to the isolated plasma membranes. This effect suggests that direct regulation of calcium homeostasis may represent an important event in the mechanism of action of insulin.

Transmembrane electrical potential and transmembrane pH gradient in the acidophile Thiobacillus ferro-oxidans.

Abstract: Thiobacillus ferro-oxidans is capable of using the oxidation of Fe2+ by O2 at pH 2.0 as the sole source of energy for growth and CO2 fixation. The bacterium maintains an intracellular pH of 6.5 over a range of external pH from 1.0 to 8.0, as measured by [14C]acetate and [3H]methylamine distribution. The membrane potential was estimated by the distribution of the lipid-soluble cation dibenzyldimethylammonium and the anion SCN-. At pH 2.0 (the pH of growth) during Fe2+ oxidation the transmembrane pH gradient is 4.5 units with an opposing membrane potential of -10mV, giving a proton electrochemical gradient of +256mV. This gradient is actively maintained.

Requirement for membrane potential in injection of phage T4 DNA.

Abstract: The first stages of infection by phage T4 may be divided into energy-dependent and energy-independent processes. Irreversible adsorption, unplugging, and initial exposure of the DNA terminus may occur at 4 degrees C, or at 37 degrees C in bacteria whose energy-yielding metabolism has been poisoned. DNA injection into the cytoplasm needs higher temperatures and energy from the host cell. The nature of this energy requirements was deduced from the use of metabolic inhibitors. Our results show that T4 DNA injection specifically requires the presence of a protonmotive force across the cytoplasmic membrane of the host. Moreover, the chemical gradient (delta pH) does not appear to be essential, but the membrane potential (delta psi) is required.

Potassium and Phosphate Uptake in Corn Roots: Further Evidence for an Electrogenic H+/K+ Exchanger and an OH−/Pi Antiporter

Abstract: Evidence is presented that K+ uptake in corn root segments is coupled to an electrogenic H+/K+ -exchanging plasmalemma ATPase while phosphate uptake is coupled to an OH−/Pi antiporter. The plasmalemma ATPase inhibitor, diethylstilbestrol, or the stimulator, fusicoccin, altered K+ uptake directly and phosphate uptake indirectly. On the other hand, mersalyl, an OH−/Pi antiporter inhibitor, inhibited phosphate uptake instantly but only slightly affected K+ uptake. Collapse of the proton gradient across the membrane by (p-trifluoromethoxy) carbonyl cyanide phenylhydrazone resulted in immediate inhibition of K+ uptake but only later inhibited phosphate uptake. Changing the pH of the absorption solution had opposite effects on K+ and phosphate uptake. In addition, a 4-hour washing of corn root tissue induced a 5-fold increase in the rate of K+ uptake with little or no lag, but only a 2- to 3-fold increase in phosphate uptake with a 30- to 45-minute lag. Collectively these differences strongly support the coupling of an electrogenic H+/K+ -exchanging ATPase to an OH−/Pi antiporter in corn root tissue.

Effect of diethylpyrocarbonate on lactose/proton symport in Escherichia coli membrane vesicles.

Abstract: Exposure of Escherichia coli ML 308-225 membrane vesicles to the histidine-specific reagent diethylpyrocarbonate (DEPC) led to concentration- and time-dependent inactivation of active lactose transport, and the sensitivity of the system to inactivation was enhanced when an electrochemical proton gradient (delta- muH+, interior negative and alkaline) was generated across the vesicle membrane. Although beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside blocked DEPC inactivation, binding of p-nitrophenyl alpha-D-galactopyranoside was not significantly altered, indicating that DEPC does not react at the binding sites of the lac carrier protein. Strikingly, vesicles treated with DEPC exhibited an increased apparent Km for delta- muH+-driven lactose transport and counterflow but no change in the Vmax of these reactions and no change in the apparent Km or Vmax of facilitated diffusion. Moreover, DEPC treatment increased the apparent Km observed for delta- muH+-driven proline and D-lactate transport with no change in Vmax. Finally, the lactose counterflow activity of DEPC-treated vesicles was regenerated by subsequent exposure to hydroxylamine. It is suggested that a histidyl residue(s) in the lac carrier or another protein in the translocation complex is involved either in the binding and translocation of protons or in a conformational change that may occur upon protonation of the lac carrier protein.

Intracellular Ionic Activities and Transmembrane Electrochemical Potential Differences in Gallbladder Epithelium

Abstract: Intracellular ion activities inNecturus gallbladder epithelium were measured with liquid ion-exchanger microelectrodes. Mean values for K, Cl and Na activities were 87, 35 and 22mm, respectively. The intracellular activities of both K and Cl are above their respective equilibrium values, whereas the Na activity is far below. This indicates that K and Cl are transported uphill toward the cell interior, whereas Na is extruded against its electrochemical gradient. The epithelium transports NaCl from mucosa to serosa. From the data presented and the known Na and Cl conductances of the cell membranes, we conclude that neutral transport driven by the Na electrochemical potential difference can account for NaCl entry at the apical membrane. At the basolateral membrane, Na is actively transported. Because of the low Cl conductance of the membrane, only a small fraction of Cl transport can be explained by diffusion. These data suggest that Cl transport across the basolateral membrane is a coupled process which involves a neutral NaCl pump, downhill KCl transport, or a Cl-anion exchange system.

Lactose carrier protein of Escherichia coli. Transport and binding of 2'-(N-dansyl)aminoethyl beta-D-thiogalactopyranoside and p-nitrophenyl alpha-d-galactopyranoside.

Abstract: The elevated level of lactose carrier protein present in cytoplasmic membranes derived from Escherichia coli strain T31RT, which carries the Y gene of the lac operon on a plasmid vector (Teather, R. M., et al. (1978) Mol. Gen. Genet. 159, 239--248), has allowed the detection of a complex between the carrier and the fluorescent substrate 2'-(N-dansyl)-aminoethyl beta-D-thiogalactopyranoside (Dns2-S-Gal). Binding is accompanied by a 50-nm blue shift in the emission maximum of the dansyl residue. The complex (dissociation constant, KD = 30 micron) rapidly dissociates upon addition of competing substrates such as beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside or upon reaction with the thiol reagent p-chloromercuribenzenesulfonate. Binding of both Dns2-S-Gal and p-nitrophenyl alpha-D-galactopyranoside (alpha-NPG) occurs spontaneously in the absence of an electrochemical potential gradient across the membrane. Comparison of equilibrium binding experiments using Dns2-S-Gal or alpha-NPG and differential labeling of the carrier with radioactive amino acids shows that the carrier binds 1 mol of substrate per mol of polypeptide (molecular weight 30 000). In addition to specific binding to the lactose carrier, Dns2-S-gal binds unspecifically to lipid vesicles or membranes, as described by a partition coefficient, K = 60, resulting in a 25-nm blue shift in the emission maximum of the dansyl group. Both Dns2-S-Gal and alpha-NPG are not only bound by the lactose carrier but also transported across the membrane by this transport protein in cells and membrane vesicles. The fluorescence changes observed with dansylated galactosides in membrane vesicles in the presence of an electrochemical gradient (Schuldiner et al. (1975) J. Biol. Chem. 250, 1361--1370)) are interpreted as an increase in unspecific binding after translocation.

Variable proton conductance of submitochondrial particles.

Abstract: The relationship between the rate of substrate oxidation and the protonmotive force (electrochemical proton gradient) generated by bovine heart submitochondrial particles has been examined Unexpectedly, oxidation of succinate generated a higher protonmotive force than the oxidation of NADH, although the rate of proton translocation across the membrane was inferred to be considerably lower with succinate as substrate The data suggest that the flow of electrons through site 1 of the respiratory chain may increase the conductance of the mitochondrial membrane for protons Upon reduction of the rate of succinate oxidation by titration with malonate, the protonmotive force remained essentially constant until the extent of inhibition was greater than 75% The general conclusion from this work is that a constant passive membrane conductance for protons cannot be assumed

Escherichia coli adenylate cyclase complex: regulation by the proton electrochemical gradient

Abstract: Sugars such as glucose are transported into Escherichia coli by a coupled phosphorylation mechanism (the phosphoenolpyruvate:sugar phosphotransferase system, PTS). Transport of sugars through the PTS results in inhibition of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity by a mechanism involving a change in the state of phosphorylation of PTS proteins. Other sugars (e.g., lactose) are transported without modification by a mechanism involving proton cotransport, which requires a proton motive force across the cell membrane. We show here that uptake of sugars through the lactose transport system results in inhibition of adenylate cyclase activity if the proton symport mechanism is also active. The protonophore carbonyl cyanide m-chlorophenylhydrazone also inhibits adenylate cyclase activity. These data suggest that the steady-state electrochemical proton gradient regulates the activity of adenylate cyclase. We propose that sugar-dependent inhibition of adenylate cyclase activity may occur by either of two mechanisms. Sugars transported by the PTS inhibited adenylate cyclase activity by dephosphorylation of a regulatory protein, while sugars transported by the proton motive force system inhibit adenylate cyclase activity as a result of collapse of the proton electrochemical gradient.

Potassium modulation of taurine transport across the frog retinal pigment epithelium.

Abstract: Net taurine transport across the frog retinal pigment epithelium-choroid was measured as a function of extracellular potassium concentration, [K+]o. The net rate of retina-to-choroid transport increased monotonically as [K+]o increased from 0.2 mM to 2 mM on the apical (neural retinal) side of the tissue. No further increase was observed when [k+]o was elevated to 5 mM. The [K+]o changes that modulate taurine transport approximate the light-induced [K+]o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium. The taurine-potassium interaction was studied by using rubidium as a substitute for potassium and measuring active rubidium transport as a function of extracellular taurine concentration. An increase in apical taurine concentration, from 0.2 mM to 2 mM, produced a threefold increase in active rubidium transport, retina to choroid. Net taurine transport can also be altered by relatively large, 55 mM, changes in [Na+]o. Apical ouabain, 10(-4) M, inhibited active taurine, rubidium, and potassium transport; in the case of taurine, this inhibition is most likely due to a decrease in the sodium electrochemical gradient. In sum, these results suggest that the apical membrane contains a taurine, sodium co-transport mechanism whose rate is modulated, indirectly, through the sodium pump. This pump has previously been shown to be electrogenic and located on the apical membrane, and its rate is modulated, indirectly, by the taurine co-transport mechanism.

Effects of external sodium and cell membrane potential on intracellular chloride activity in gallbladder epithelium.

Abstract: Conventional and Cl-selective liquid ion-exchanger intracellular microelectrodes were employed to study the effects of extracellular ionic substitutions on intracellular Cl activity (aCli) in Necturus gallbladder epithelium. As shown previously (Reuss, L., Weinman, S.A., 1979; J. Membrane Biol. 49:345), when the tissue was exposed to NaCl-Ringer on both sides aCli was about 30 mM, i.e., much higher than the activity predicted from equilibrium distribution (aCleq) across either membrane (5--9 mM). Removal of Cl from the apical side caused a reversible decrease of aCli towards the equilibrium value across the basolateral membrane. A new steady-state aCli was reached in about 10 min. Removal of Na from the mucosal medium or from both media also caused reversible decreases of aCli when Li, choline, tetramethylammonium or N-methyl-D-glucamine (NMDG) were employed to replace Na. During bilateral Na substitutions with choline the cells depolarized significantly. However, no change of cell potential was observed when NMDG was employed as Na substitute. Na replacements with choline or NMDG on the serosal side only did not change aCli. When K substituted for mucosal Na, the cells depolarized and aCli rose significantly. Combinations of K for Na and Cl for SO4 substitutions showed that net Cl entry during cell depolarization can take place across either membrane. The increase of aCli in depolarized cells exposed to K2SO4-Ringer on the mucosal side indicates that the basolateral membrane Cl permeability (PCl) increased. These results support the hypothesis that NaCl entry at the apical membrane occurs by an electroneutral mechanism, driven by the Na electrochemical gradient. In addition, we suggest that Cl entry during cell depolarization is downhill and involves an increase of basolateral membrane PCl.

Anaerobic Respiration and Energy Conservation in Paracoccus denitrficans

Abstract: 1 Electron paramagnetic resonance spectra at 8–60 K of NADH-reduced membrane particles prepared from Paracoccus denitrificans grown anaerobically with nitrate as terminal electron acceptor show the presence of iron-sulfur centers 1–4 in the NADH-ubiquinone segment of the respiratory chain. In addition resonance lines at g= 2.058, g= 1.953 and g= 1.88 are detectable in the spectra of succinate-reduced membranes at 15 K, which are attributed to the iron-sulfur-containing nitrate reductase. 2 Sulphate-limited growth under anaerobic conditions does not affect the iron-sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain-linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate-limited growth of P. denitrificans under aerobic conditions [Eur. J. Biochem. (1977) 81, 267–275]. 3 Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain-linked energy conservation observed during nitrate respiration [Arch. Microbiol. (1977) 112, 17–23] can be explained by the uncoupling action of nitrite.

Stimulus-secretion coupling of glucose-induced insulin release. XXIX. Regulation of 86Rb+ efflux from perfused islets.

Abstract: Glucose provokes a dose-related, rapid, sustained, and rapidly reversible reduction in the fractional outflow rate of 86Rb+ from perfused pancreatic islets. This efflux probably corresponds to a passive movement driven by the electrochemical gradient of K+ across the plasma membrane and mediated by a native ionphoretic system. Indeed, it is facilitated by valinomycin or cell membrane depolarization, little affected by ouabain, and inhibited by verapamil or omission of extracellular K+. The effect of glucose upon 86Rb+ efflux does not appear to be directly attributable to changes in either glucose transport, plasma cell polarization, Na+ influx, cyclic AMP concentration, or insulin secretion. Although a modulatory role of intracellular Ca2+ on K+ conductance cannot be ruled out, the experimental data suggest rather that the glucose-induced modification of 86Rb+ fractional outflow rate is directly linked, for its major part, to metabolic events such as an increase in the rate of glycolysis and/or generation of reduced pyridine nucleotides.

Control of phosphoenolpyruvate-dependent phosphotransferase-mediated sugar transport in Escherichia coli by energization of the cell membrane.

Abstract: The phosphoenolpyruvate-dependent phosphotransferase-mediated sugar transport in Escherichia coli is inhibited by the energized of the membrane. This was shown in intact cells as well as in membrane vesicles. Relaxation of the proton gradient by uncouplers stimulated the uptake of sugars via the phosphotransferase system in aerobically cultured cells. No such effect was seen in anaerobic cells, apparently because the cell membrane of these cells is poorly energized. Energization by respiration of D-lactate or ascorbate inhibited the phosphotransferase uptake system in membrane vesicles. This inhibition was reversed by the addition of cyanide. Oxamate, a specific inhibitor of lactate dehydrogenase, prevented the inhibitory effect of D-lactate. Membrane vesicles prepared from a cytochrome-less mutant were not energized by D-lactate oxidation and the phosphotransferase uptake system was not inhibited.

Membrane potentials and sugar transport by ATP-depleted intestinal cells: effect of anion gradients.

Abstract: Rotenone or dinitrophenol treatment was used to decrease cellular ATP levels in isolated chicken intestinal epithelial cells by over 90% and to produce a cell population in which no steady-state ac...

Control of electron flow in intact chloroplasts by the intrathylakoid pH, not by the phosphorylation potential.

Abstract: In the presence of nitrite or oxaloacetate, intact chloroplasts evolved oxygen at a significant rate for the initial 1 to 2 min of illumination. Subsequently, oxygen evolution was suppressed progressively. The suppressed oxygen evolution was stimulated strikingly by NH4Cl. The results indicate that coupled electron flow in intact chloroplasts is controlled in the light, and the control is released by NH4Cl. However, at low concentrations, NH4Cl was not an effective uncoupler of photophosphorylation in intact chloroplasts. Intrachloroplast ATP levels and ATP/ADP ratios were not significantly influenced by NH4Cl. In contrast, the quenching of 9-aminoacridine fluorescence, which can be used to indicate the intrathylakoid pH in intact chloroplasts, was reduced drastically even by low concentrations of NH4Cl. This suggests that the chloroplast phosphorylation potential is not in equilibrium with the proton gradient. In coupled chloroplasts, the intrathylakoid pH was lower in the light with nitrite than with oxaloacetate as electron acceptor. Electron flow was also more effectively controlled in chloroplasts illuminated with nitrite than with oxaloacetate. It is concluded that the intrathylakoid pH, not the phosphorylation potential, is a factor in the control of the rate of electron flow in intact chloroplasts.