Formation Of pH Gradient Research Articles

Optical measurements of ΔpH and Δψ in corn root membrne vesicles: Kinetic analysis of Cl− effects on a proton-translocating ATPase

The optical probes 9-aminoacridine, quinacrine, and bis (3-propyl-5-oxoisoxazol-4-yl) pentamethine oxonol (OX-VI) were used to measure the formation of pH and electric potential gradients in vesicles believed to be derived from corn root tonoplast membranes. Under certain conditions the quenching of fluorescence of 9-aminoacridine was shown to be quantitatively related to ΔpH, and the shift in the absorbance spectrum (measured as absorbance difference at 610 and 580 nm) of OX-VI shown to be quantitatively related to Δψ. In the absence of chloride ions, a positive interior membrane potential of approximately +100 mV was formed upon the addition of 5mm Mg/ATP to a suspension of membrane vesicles. The addition of Cl− salts reduced Δψ and stimulated the formation of ΔpH. In the presence of 50mmCl−, a ΔpH of approximately 1.1 units was established following the addition of 5mm Mg/ATP. The kinetics of Cl− activation of H+ transport could be resolved into a linear and a saturable component, with aKm for the saturable component between 4 and 5mm. Cl− inhibition of Δψ showed similar kinetics, indicating that Cl− activates electrogenic H+ transport as a permeant anion. The biphasic kinetics suggest 4 two pathways for Cl\t- permeation, with the linear component attributable to permeation through the lipid bilayer and the saturable component attributable to permeation through an anion channel. This model is supported by the effects of the anion channel blockers, 4-acetamido-4\t'-isothiocyano-2,2\t'-stilbenedisulfonic acid (SITS) and 4,4\t'-diisothiocyano-2,2\t'-stilbenedisulfonic acid (DIDS), which abolished the saturable component but not the linear component of Cl\t- stimulated H+ transport.

Formation of natural pH gradients in sequential moving boundary systems with solvent counterions II. Predicted and experimental properties

AbstractSequential moving boundary electrophoresis with protons and hydroxyl ions as the sole counterions, consisting solely of six acids (system A) and of six bases (system B), gives rise to natural steady‐state pH gradients similar to those predicted by theory (pH 2.4–5.5 for system A, pH 10.3–12.2 for system B). Mixtures of the constituents in system A and system B gave rise to a natural pH gradient spanning the pH range between the terminal electrolytes (pH 2.4 and 12.2) predicted for systems A and B. The predicted boundary displacement is negligibly small. Experimentally the boundary displacement could not be determined in view of the difficulty of separating it from the initial transient state in which the pH gradient is formed, and a final state in which the pH gradient decays.

Properties of narrow-range ampholytes isolated from wide-range ampholyte preparations

A simple procedure for obtaining useful narrow-pH-range ampholytes from inexpensive laboratory-synthesized ampholytes by preparative isoelectric focusing in Pevikon is described. The narrow range ampholytes prepared in this way are comparable to commercial ampholyte preparation as judged by conductivity, buffer capacity, pH gradient formation, and resolving power. These inexpensive narrow-range ampholytes are particularly well suited to preparative isoelectric focusing applications requiring large quantities of ampholytes.

Growth of Bacillus cereus in a Gel-stabilized Nutrient Gradient System

Bacillus cereus consistently formed multiple growth bands in gel-stabilized gradient systems. The development of inoculated and uninoculated systems was followed by gel scanning and by measuring physico-chemical parameters. Factors which influenced band formation included the availability of oxygen and the presence of a glucose gradient. The relative position of bands was dependent on the glucose concentration in the source layer and the strength of the basal medium. Other factors such as agar type and concentration were relatively unimportant. The generation of steep pH gradients in the gel profoundly affected band formation. Thus, in the presence of a high buffering capacity changes in band pattern and pH profile occurred. No bands and no pH gradient were observed in gels incubated anaerobically. Addition of an alkaline overlayer, however, resulted in a pH gradient and band formation. Although pH gradients were responsible for sub-surface bands, it was not clear what caused the ‘chopping’ of growth into discrete bands. Computer simulations suggested that an asymmetric activation threshold may be responsible, and the mechanism may involve sporulation and germination.

Isolation and assay of corn root membrane vesicles with reduced proton permeability

Conditions promoting the formation of sealed membrane vesicles from corn roots with reduced proton permeability were examined using the probe 9-aminoacridine as a rapid indicator of pH gradient formation and dissipation. Plasma membrane vesicles isolated by differential and density gradient centrifugation were leaky to protons and rapidly equilibrated when exposed to artificially imposed pH gradients. The leaky plasma membrane vesicles showed reduced proton permeability when incubated with calcium or with excess phospholipids. However, these vesicles were unable to form ATP-induced pH gradients. Sealed vesicles isolated by discontinuous Ficoll gradient centrifugation of a microsomal fraction displayed reduced proton permeability and were osmotically active. In contrast to purified plasma membrane vesicles, the microsomal-derived vesicles were more suitable for studies of active proton transport.

Intact chloroplast electron flow. Effects of ribose 5-phosphate

Additions of ribose 5-phosphate to intact spinach chloroplasts were used to probe the effects of ADP regeneration on pH-gradient formation and electron-transfer reactions. In weakly illuminated chloroplasts, the ATP ADP ratio dropped by 64% and the transthylakoid pH gradient decreased by a minimum of 0.2 units in response to ribose 5-phosphate. Nitrite reduction increased 2-fold while, under conditions of cyclic electron flow, the half-time for cytochrome f reduction decreased by a factor of two from 4.1 to 1.9 ms. The results suggest that metabolic ATP consumption, during the conversion of ribulose 5-phosphate to ribulose 1,5-bisphosphate, enhances electron transfer between plastohydroquinone and cytochrome f through decreases in the transthylakoid pH gradient caused by phosphorylation of ADP.

Kinetics of glutamine efflux from liver mitochondria loaded with the 14C-labeled substrate

Glutamine transport across the inner membrane of rat liver mitochondria was studied by the method of loading the organelles with [ 14C]glutamine and by measuring efflux of the metabolite at 0°C. The release of [ 14C]glutamine from loaded mitochondria was prevented by mersalyl, whereas the efflux was started by the addition of glutathione. The rate of glutamine efflux from the mitochondria was measured by the inhibitor stop technique with mersalyl plus N- ethylmaleimide . It was found that up to 10 mM glutamine there is no significant activity of glutaminase, whereas at about 20 mM of the substrate the enzyme is activated. The rate of the efflux measured after the addition of the optimal amount of glutathione was 10 nmol glutamine/min per mg protein. This is 5-times faster than the rate of glutaminase activity at 0°C. The pH optimum of glutamine carrier is between 6.5 and 7.0. Low concentration of succinate inhibits the efflux due to formation of pH gradient in coupled mitochondria, whereas a higher concentration of succinate inhibits the carrier directly. 2-Oxoglutarate and glutamate strongly inhibit the rate of glutamine efflux, the inhibition by glutamate being very pronounced at its physiological concentration. d-Glutamine does not inhibit the rate of the efflux, indicating that the transport of l-glutamine is stereospecific.

DCCD inhibits proton translocation and electron flow at the second site of the mitochondrial respiratory chain

DCCD inhibits formation of a succinate-driven transmembrane pH gradient in submitochondrial particles, as shown by inhibition of fluorescence quenching of 9-aminoacridine, without concomitant inhibition of succinate oxidation. On the other hand ubiquinol-cytochrome c reductase activity is inhibited by DCCD. Half-inhibition of both fluorescence quenching and ubiquinol-cytochrome c reductase occur at 35 μM DCCD. The results suggest that DCCD inhibits proton pumping activity coupled to electron flow through the bc 1 complex.

Buffer-focusing chromatography using multicomponent electrolyte elution systems

Procedures for the generation of internal pH gradients on columns of ion-exchange resins using multicomponent eluent systems of defined chemical composition, comprised of common amphoteric and non-amphoteric buffers, are described. The influence of buffer concentration, pH and resin characteristics on pH gradient formation have been examined. The physicochemical basis of the pH gradient formation under these chromatographic conditions has been discussed in terms of dissociation theory for polyelectrolytes which highlights the similarity with the formation of natural pH gradients by buffer electrofocusing procedures. Applications of this chromatographic technique for the separation of proteins are described.

Proton gradients in renal cortex brush-border membrane vesicles. Demonstration of a rheogenic proton flux with acridine orange.

The fluorescence quenching of acridine orange has been used to study the formation and dissipation of acid interior pH gradients in brush-border membrane vesicles from rabbit renal cortex. Acidic interior pH gradients were produced by 1) outwardly directed gradients of Na+ or K+, and 2) the addition of vesicles equilibrated at pH 6.0 to 7.5 buffer. The rate of pH gradient dissipation was stimulated 6.3-fold by the replacement of tetramethylammonium gluconate by tetramethylammonium chloride. A further increase, of 2-fold, was seen upon the addition of carbonyl cyanide-m-chlorophenylhydrazine, demonstrating the existence of a Cl- conductance pathway. In the presence of valinomycin, the replacement of tetramethylammonium gluconate by K gluconate increased the rate of delta pH dissipation by 11-fold, demonstrating the existence of a conductive pathway for protons. This pathway for protons was also shown by the formation of an acidic interior space by an outwardly directed K gradient in the presence of valinomycin. The parallel conductive pathways for H+ and Cl- may dissipate pH and chloride gradients across the luminal membrane of the proximal tubule.

SUBSTRATE ENTRAPMENT AND FORMATION OF pH GRADIENT IN AMMONIUM BILAYER VESICLES

Abstract Trapping of water-soluble amines in the inner water core of ammonium bilayer vesicles was determined by reaction with fluorescamine, and the formation of pH gradient was confirmed by fluorescence quenching of membrane-bound riboflavin.

Thallium interaction with the gastric (K, H)-ATPase.

The gastric (K, H)-ATPase has been shown to catalyze an electroneutral H+ for K+ exchange. Tl+ is able to substitute for K+ as an activating cation in the hydrolytic reaction with an apparent dissociation constant of 90 microM as compared to about 870 microM for K+. The ability of Tl+ to participate in transport is shown by the development of pH gradients in the presence of Tl+ following addition of ATP to gastric vesicles and by the ATP-dependent efflux of Tl+ from gastric vesicles. Inhibition of hydrolysis is observed at pH 7.4 with external Tl+ concentrations above 3.0 mM. This inhibition of hydrolysis is correlated with inhibition of pH-gradient formation. The inhibition of transport activity is partially relieved by a decrease in medium pH. This inhibitory effect is attributed to Tl+ binding at an external, low affinity cation site. In contrast to rubidium chloride, at high Tl+ concentrations, following the initial Tl+ efflux, there is reuptake of the cation. This rapid uptake is attributed to lipid-dependent Tl+ entry pathways. The vesicles exhibit a high permeability to thallium nitrate demonstrating a half-time (t1/2) for uptake of about 1.0 min in contrast to 46 min for rubidium chloride. In both gastric vesicles or liposomes, external Tl+ concentrations in excess of 1 to 4 mM are able to dissipate intravesicular proton gradients. Thus, although Tl+ is able to activate the gastric ATPase by mimicking K+, the permeability of this cation in lipid bilayers tends to uncouple H+ transport at concentrations high enough to generate detectable proton gradients.

THE CONTRIBUTION OF RHODOSPZRZLLUM RUBRUM FERREDOXIN II TO IN VIVO EPR SIGNALS AND ITS ROLE IN ELECTRON TRANSPORT

Abstract—Evidence is presented that Fd II, an iron‐sulfur protein containing 4 irons and 4 acid‐labile sulfurs, is responsible for a number of signals previously reported detectable in cells of R. rubrum. When oxidized, Fd II exhibits a g = 2.012 EPR signal which is readily detected in R. rubrum cells. In our hands, Fd II is photochemically reduced to an EPR‐silent product contrary to the results of other investigators. However. in the presence of reducing agents. the reduced form is apparently denatured upon freezing. The denatured form exhibits EPR signals similar to some also previously observed in whole cells.Fd II catalyzes the ascorbate‐DCPIP linked photoreduction of NAD with R. rubrum chromatophores even in the presence of an inhibitor which suppresses the formation of pH and emf gradients. This result raises the possibility of a role for Fd II in non‐cyclic electron transport in R. rubrum.

Coexistence of steady state and transient state in isoelectric focusing

The possibility of transforming a wide (pH 3.5–10) into a narrow pH gradient in isoelectric focusing (IEF) has been examined by the use of different amino acid terminators at the electrons, viz 50 m M Asp, 50 m M Trp, 45 m M Phe. 0.5 M Gly or 0.5 M Lys. The ph gradient formation and decay seem to be insensitive to the type of amino acid used or to its molarity. While Ampholine and protein samples reach equilibrium positions, large amounts of amino acids move as two wave fronts, from the anode and cathode, unsuccessfully trying to reach their p I positions. Thus, the steady state and transient state coexist in IEF and are quite insensitive to each other. The formation and stabilization of the pH gradient in IEF do not require a performed “pH-cage”, nor can IEF be regarded as isotachophoresis, since the same ion acts simultaneously as the “leading” and “terminating” ion in our system. When the same buffer is used simultaneously at the anode and cathode, the gel loses its polarity so that the system becomes insensitive to the choice of electrode position, or to polarity reversal at any time during the IEF experiment.

Energy transduction in Escherichia coli: new mutation affecting the Fo portion of the ATP synthetase complex.

A mutation affecting the intrinsic membrane portion (BFo) of the ATP synthetase complex is described. The phenotype is different from previously reported BFo mutants. This mutation results in the ability of membranes lacking the extrinsic membrane portion (BF1) of the ATP synthetase complex to maintain a transmembrane pH gradient. Unlike other BFo mutants, this strain, NR71, is capable of utilizing ATP hydrolysis for the formation of a transmembrane pH gradient.

Visualization of protein zones in preparative electrophoresis and carrier ampholyte zones on isoelectric focusing in gel slabs by two light refraction methods

Two simple arrangements for the detection of refractive index variations are described. One is based on the so-called shadow method and consists of a lamp, a parabolic mirror, and a screen. The other is based on the Toepler-schlieren method and consists of a lamp, two parabolic mirrors, an adjustable knife-edge, and a screen. These devices are used to visualize refractive index variations in a vertical polyacrylamide-gel slab used for electrophoretic separations. The variations appear on a screen as differences in illumination in a natural-size image of the gel. The electrophoresis apparatus is made of ordinary window glass and acrylic plastic. Two experiments are described: visualization of protein zones during electrophoresis and carrier ampholyte zones during isoelectric focusing. Two major applications are suggested: localization of protein zones to guide proper slicing of the gel in preparative work (for analytical purposes, more sensitive methods are necessary) and monitoring of the formation of pH gradients.

Changes in Internal Hydrogen Ion Concentration Associated with Photophosphorylation in Intact and Sonically Treated Chloroplasts

Abstract By using different pH indicators it is possible to demonstrate, in the presence or absence of phosphorylation, the existence of two compartments in chloroplasts, one of which becomes more acidic and the other more basic during electron flow. The observed magnitude of the pH differential between these two compartments is over 1.5 pH units. Cations, such as NH4+, N-methylphenazonium methosulfate, and protonated neutral red, which compete with H+ ions for entry into chloroplasts, inhibit the development of pH gradients within chloroplasts. Diffusible anions, such as acetate and orthophosphate, also block formation of these internal pH gradients. All of the above inhibitors of formation of internal pH gradients also inhibit phosphorylation, in a parallel manner. Sonic treatment of chloroplasts causes progressive and parallel inhibition of phosphorylation and formation of internal pH gradients. These data support the concept that chloroplasts contain a proton-translocating oxidation-reduction chain and that the resultant efflux of H+ ions through chloroplast membranes supplies the energy for phosphorylation.