Passive potassium ion permeability of Halobacterium halobium cell envelope membranes.

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

Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 10⁴ to 12.8 · 10⁴ M^?. s^?1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s^?1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside with concanavalin A were examined. The value of the binding constant K_ap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression K_ap = K_aK(K_a + [H⁺])^?1. The values of K_a, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10^?4 M and 1 · 10^?4 M for 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl α-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α-D-mannopyranoside to concanavalin A at pH 5.2 and 25°C.     

Protective effect of EGTA on rat gastric membranes enriched in (H+-K+)-ATPase

Rat gastric membranes enriched in (H⁺-K⁺)-ATPase, when prepared in the presence of 1 mM ethyleneglycol-bis-(β-aminoethyl ether)N,N′-tetraacetic acid, showed the ability to accumulate H⁺ ions upon addition of ATP, KCl, and valinomycin. The membranes were largely impermeable to K⁺ and Cl^?. In contrast, the rat membranes prepared without the Ca²⁺ chelator lost the ability to develop a pH gradient because of the membrane leakiness to H⁺. A majority of these membrane vesicles became also permeable to K⁺. We suggest that the calcium chelator preserved the gastric membrane permeability barrier during isolation by inhibiting various Ca²⁺-dependent phospholipases in rat gastric mucosa.     

Kinetics of cation-induced aggregation of Torpedo electric organ synaptic vesicles

Synaptic vesicles from the Torpedo ray can be induced to aggregate in the presence of Ca²⁺ and K⁺ in the 4 mM and 50 mM range, respectively. The reactions are strikingly similar to those of chromaffin granule membranes reported previously (Morris, S.J., Chiu, V.C.K. and Haynes, D.H. (1979) Membrane Biochem. 2, 163–202). The Ca²⁺-induced reaction includes dimerization and higher order aggregation, and is shown to be due to electrostatic screening interactions and binding to negatively-charged groups on the membrane surface. The K⁺-induced reaction includes only dimerization and is shown to be due to screening interactions alone.The kinetics of the dimerization reactions were studied using the stopped-flow rapid mixing technique. The Ca²⁺-induced reaction has a ‘bimolecular’ rate constant of 4.77 · 10⁸ M^?1 · s^?1 while the value for the K⁺-induced reaction is 7.05 · 10⁹ M^?1 · s^?1. These values are close to the limit of diffusion control (8.03 · 10⁹ M^?1 · s^?1), indicating that no large energy barriers or structural barriers to aggregation exist. Arrhenius plots for the Ca²⁺-induced aggregation showed a break at 5°C. Above this temperature, the activation energy is low ($\text{+0.65}\text{kcal/mol}$), consistent with the above. Below this temperature, the activation energy is high, consistent with a membrane structure change increasing the energetic and structural barriers. This information, and the observation of a high stability constant of the complex, were taken as evidence for the involvement of ‘recognition sites’ on the membrane surface.     

Relationship between proton motive force and potassium ion transport in Halobacterium halobium envelope vesicles.

Membrane vesicles prepared from Halobacterium halobium extrude protons during illumination, and a pH difference (inside alkaline) and an electrical potential (inside negative) develop. The sizes of these gradients and their relative magnitudes are dependent on a complex interaction among the proton-pumping activity of bacteriorhodopsin, Na⁺ extrusion through an antiport system, and the ability of K⁺ and Cl^? to act as counterions to the electrogenic movement of H⁺. The net result of these variable effects is that the electrical potential is relatively independent of external pH, whereas the pH difference tends toward zero when the pH is increased to 7.5–8. Although the light-induced pH difference is greater in KCl than in NaCl, and the electrical potential smaller, this is not caused by a high permeability of the vesicle membranes to K⁺. The vesicle membrane is poorly permeable to K⁺, as shown by: lack of a K⁺ diffusion potential in the absence of valinomycin, light-induced electrical potentials which are in excess of the chemical potential difference for K⁺, and direct measurements of the slow rate of K⁺ influx during illumination. The finding that the rate of K⁺ uptake is a linear function of external K⁺ concentration between 0 and 1 m is inconsistent with the existence of a specific K⁺ permeation mechanism in these vesicles. Since at external K⁺ concentrations < 1.4 m the extrusion of Na⁺ during illumination proceeds much more rapidly than K⁺ influx, it must be concluded that the vesicles also lose Cl^? and water. Measurements of light-scattering changes confirm that under these conditions the vesicles collapse. The light-induced collapse is diminished only when the inward movement of K⁺ is increased, either by increasing the external K⁺ concentration or by adding valinomycin.     

A thermally induced alteration in lysosome membranes: Salt permeability at 0 and 37 °C

Preparations of radioactive lysosomes were obtained from mouse kidney after injection of radioactive iodine-labeled bovine ribonuclease. Stability of these lysosomes in various media was estimated from measurements of proteolytic activity towards the ribonuclease, and of ribonuclease retention in particles. The lysosomes were stable at 37 °C in isotonic, sucrose-free solutions of KCl, NaCl and potassium acetate, and in mixtures of these with MgCl₂, showing that these salts are relatively impermeant through the lysosomal membranes. The membranes were less permeable to Na⁺ than to K⁺. Both KCl and NaCl exerted their optimal protective effects over a broad concentration range above 0.125 M in 0.025 M acetate buffer. Mg²⁺ enhanced the protective effect of both K⁺ and Na⁺; the osmotic effect of 0.075 M NaCl-0.05 M MgCl₂ was indistinguishable during the entire course of ribonuclease digestion from that of isotonic sucrose. Osmotic protection by KCl-MgCl₂ was demonstrated over the pH range 5.5–7.0. A marked alteration in membrane properties occurs at lower temperatures in 0.11 M KCl-0.01 M MgCl₂ such that, at 0 °C, K⁺ permeability is much higher than at 37 °C, as shown by a several-fold decrease in stability at the lower temperature.     

Equilibrium constants under physiological conditions for the reactions of the nonphosphorylated pathway of L-serine biosynthesis

The observed equilibrium constants (K_obs) for the reactions of d-2-phosphoglycerate phosphatase, d-2-Phosphoglycerate^3? + H₂O → d-glycerate^? + HPO₄^2?; d-glycerate dehydrogenase (EC 1.1.1.29), d-Glycerate^? + NAD⁺ → NADH + hydroxypyruvate^? + H⁺; and l-serine:pyruvate aminotransferase (EC 2.6.1.51), Hydroxypyruvate^? + l-H · alanine^± → pyruvate^? + l-H · serine^±; have been determined, directly and indirectly, at 38 °C and under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal-binding constants of the substrates, an ionic equilibrium constant (K) also has been calculated for each reaction. The value of K for the d-2-phosphoglycerate phosphatase reaction is 4.00 × 10³m [ΔG⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)]([H₂0] = 1). Values of K_obs for this reaction at 38 °C, [K⁺] = 0.2 m, I = 0.25 M, and pH 7.0 include 3.39 × 10³m (free [Mg²⁺] = 0), 3.23 × 10³m (free [Mg²⁺] = 10^?3m), and 2.32 × 10³m (free [Mg²⁺] = 10^?2m). The value of K for the d-glycerate dehydrogenase reaction has been determined to be 4.36 ± 0.13 × 10^?13m (38 °C, I = 0.25 M) [ΔG⁰ = 73.6 kJ/mol (17.6 kcal/mol)]. This constant is relatively insensitive to free magnesium concentrations but is affected by changes in temperature [ΔH⁰ = 46.9 kJ/mol (11.2 kcal/mol)]. The value of K for the serine:pyruvate aminotransferase reaction is 5.41 ± 0.11 [ΔG⁰ = ?4.37 kJ/mol (?1.04 kcal/mol)] at 38 °C (I = 0.25 M) and shows a small temperature effect [ΔH⁰ = 16.3 kJ/ mol (3.9 kcal/mol)]. The constant showed no significant effect of ionic strength (0.06–1.0 m) and a response to the hydrogen ion concentration only above pH 8.5. The value of K_obs is 5.50 ± 0.11 at pH 7.0 (38 °C, [K⁺] = 0.2 m, [Mg²⁺] = 0, I = 0.25 M). The results have also allowed the value of K for the d-glycerate kinase reaction (EC 2.7.1.31), d-Glycerate^? + ATP^4? → d-2-phosphoglycerate^3? + ADP^3? + H⁺, to be calculated to be 32.5 m (38 °C, I = 0.25 M). Values for K_obs for this reaction under these conditions and at pH 7.0 include 236 (free [Mg²⁺] = 0) and 50.8 (free [Mg²⁺] = 10^?3m).     

Biosynthèse de l'udpglucose par les microsomes des hépatocytes de ratBiosynthesis of UDPglucose by rat liver microsomes

Evidence is presented to show that all enzymes and all intermediary metabolites of a UDPglucose biosynthesis pathway are present in the microsomal membranes of rat liver. Glucose 6-phosphate, glucose 1-phosphate and UDPglucose are characterized by chromatography.The properties of phosphoglucomutase and UTP: D-Glucose-1-phosphate uridyltransferase are studied. The K_m values of phosphoglucomutase at pH 7.2 and 42°C were 0.26 · 10^?3 mM for glucose 1,6-diphosphate and 80 · 10^?3 mM for glucose 1-phosphate. The K_m values of UTP: D-glucose-1-phosphate uridyltransferase at pH 8.5 and 37°C were 220 · 10^?3 mM for UTP and 166 · 10^?3 mM for glucose 1-phosphate. These values are compared to the given values for enzymes from different species, and to those found for soluble enzymes. The significance of this membranous pathway is discussed.     

Superoxide dismutase: a photochemical augmentation assay.

Cell envelope vesicles containing bacteriorhodopsin, prepared from Halobacterium halobium, have previously been shown to accumulate glutamate to high concentration gradients when illuminated. This active transport is energized by a sodium gradient (Na_out⁺ ? Na_in⁺), which arises from Na⁺-efflux coupled to the light-induced H⁺-gradient. The oxidation of dimethyl phenylenediamine (DPD) by the vesicles also can drive uphill glutamate transport, and such transport is inhibited by KCN, azide, ionophores, or uncouplers. K_T for glutamate is 1.4 × 10^?7m under these conditions, as compared to 1.3 × 10^?7m for light-induced transport. The respiration-induced transport of glutamate is dependent on high Na⁺ concentrations on the vesicle exterior and requires low Na⁺ concentrations in the interior. When Na⁺ of increasing concentrations is included in the vesicles, transport proceeds with increasing lags, similarly to the case of light-driven transport. In vesicles to which DPD is added first, and then KCN at increasing time intervals (5 to 15 min), glutamate transport occurs after the addition of KCN, with increasing rates, even though respiration is inhibited. This indicates that the energy generated by DPD-oxidation is conserved over several minutes. These results suggest that in the case of respiration-dependent glutamate transport the translocation is also driven by a Na⁺-gradient; thus, there is a single glutamate transport system independent of the source of energy. The generation of such an Na⁺-gradient during DPD-oxidation implies that the respiration component involved, cytochrome oxidase, is functionally equivalent to bacteriorhodopsin, which acts as a proton pump.     

A study of the current-voltage relationships of electrogenetic active and passive membrane elements in riccia fluitans

Potassium- and proton-dependent membrane potential, conductance, and current-voltage characteristics (I–V curves) have been measured on rhizoid cells of the liverwort Riccia fluitans. The potential difference (E_m) measured with microelectrodes across plasmalemma and tonoplast is depolarized to the potassium-sensitive diffusion potential (E_D) in the presence of 1 mM NaCN, 1 mM NaN₃, or at temperatures below 6°C. Whereas the temperature change from 25°C to 5°C decreases the membrane conductance (g_m) from 0.71 to 0.43 S ? m^?2, 1 mM NaCN increases g_m by about 25%. The membrane displays potassium-controlled rectification which gradually disappears at temperatures below 5°C. The potassium pathway can be described by an equivalent circuit of a diode and an ohmic resistor in parallel. In the potential interval of E_D ± 100 mV the measured I-V curves roughly fit the theoretical curves obtained from a modified diode equation. ⁸⁶Rb⁺(K⁺)-influx is voltage sensitive: In the presence of 1 mM NaCN, ⁸⁶Rb⁺-influx follows a hyperbolic function corresponding to a low conductance at low [K⁺]_o and high conductance at high [K⁺]_o. On the contrary ⁸⁶Rb⁺-influx is linear with [K⁺]_o when pump activity is normal. It is believed that there are two K⁺-transport pathways in the Riccia membrane, one of which is assigned to the low conductance (0.2 S · m^?2), the other to a temperature-dependent facilitated diffusion system with a higher conductance (7.7 S · m^?2). The electrogenic pump essentially acts as a current source and consumes about 39% of the cellular ATP-turnover. In the presence of 30 μM CCCP the saturation current of 0.1 A · m^?2 is doubled to about 0.2 A · m^?2, and the electromotive force of ?360 mV switches to ?250 mV. It is suggested that this may be due to a change in stoichiometry from one to two transported charges per ATP hydrolyzed.     

The effect of pressure and the volume of activation on the monovalent cation and glucose permeabilities of liposomes of varying composition

Measurement of the cation permeability of phospholipid microvesicles as function of pressure confirmed that a single rate-determining step occured in each case.The volume of activation was 20 ml · mole^?1 for Na⁺ and K⁺, and about 40 ml · mole^?1 for valinomycin-mediated K⁺ permeability. It was virtually independent of membrane composition. The results were explained in terms of Träuble's theory of kink-substrate dissociation at the membrane interface involving possible 2g 1 and 2g 2 kink isomers.The volume of activation for d-glucose was 37 ml · mole^?1, which was not significantly different from that for any of the valinomycin-mediated K⁺ permeabilities. However, other data suggest that the rate-limiting steps for the sugar and cation permeabilities are not the same.     

Effect of external ATP on the plasma membrane permeability and (Na++K+)-ATPase activity of mouse neuroblastoma cells

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na⁺ relative to K⁺, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K⁺ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na⁺. Under these conditions the pumping activity of the Neuro-2A (Na⁺+K⁺)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na⁺+K⁺)-ATPase for ouabain, as measured by direct [³H]ouabain-binding studies and by inhibition studies of active K⁺ uptake. In the presence of 3.5 mM ATP and the absence of external K⁺ both techniques indicate an apparent dissociation constant for ouabain of 2·10^?6 M. Neuro-2A cells contain (3.5±0.7)·10⁵ ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7±0.4)·10^?20 mol K⁺/min per copy of (Na⁺+K⁺)-ATPase at room temperature.     

Production,characteristics and applications of phytase from a rhizosphere isolated <Emphasis Type="Italic">Enterobacter</Emphasis> sp. ACSS

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40–80 °C) and pH (2.0–6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m of the pure phytase were 0.21 mM, 131.58 nmol mg^?1 s^?1, 1.64 × 10³ s^?1, and 7.81 × 10⁶ M^?1 s^?1, respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca⁺², Mg⁺² and Mn⁺², but inhibited by Zn⁺², Cu⁺², Fe⁺², Pb⁺², Ba⁺² and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.     

Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

The MgATP-stimulated accumulation of (-)-³H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K⁺ concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)-³ H-NE (Km 1 × 10^?6 M) in this system (37°) was examined under saturating (10^?5 M) and non-saturating (2 × 10^?7 M) concentrations of NE. At 10^?5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated $\text{in}$$\text{vivo}$) and 6.0±0.79 (without MgATP). At 2×10^?7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2x10^?7 M).     

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.     

Voltage dependent potassium fluxes and the significance of action potentials in Acetabularia

Membrane potential, V_m, and K⁺(⁸⁶Rb⁺) fluxes have been measured simultaneously on individual cells of Acetabularia mediterranea. During resting state (resting potential approx. ?170 mV) the K⁺ influx amounts to 0.24–0.6 pmol · cm^?2 · s^?1 and the K⁺ efflux to 0.2–1.5 pmol · cm^?2 s^?1. According to the K⁺ concentrations inside and outside the cell (40 : 1) the voltage dependent K⁺ flux (zero at V_m = E_K = ?90 mV) is stimulated approx. 40-fold for V_m more positive than E_K.It is calculated that during one action potential (temporary depolarization to V_m more positive than E_K) a cell looses the same amount of K⁺, which leaks in during 10–20 min in the resting state (V_m = ?170 mV). Since action potentials occur spontaneously in Acetabularia, they are therefore suggested to have a significant function for the K⁺ balance of this alga.     

Proton transport by gastric membrane vesicles

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H⁺ uptake into an osmotically sensitive space. The apparent K_m for ATP was 1.7 · 10^?4 M in the absence of a K⁺ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺ and is inhibited by ATPase inhibitors such as N,N′-dicylclohexylcarbodiimide. Maximal H⁺ uptake occurs with an outward K⁺ gradient but the minimal apparent K_A is found in the absence of a K⁺ gradient. The pH optimum for H⁺ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphorylation of the enzyme, but is considerably less than the pH maximum of the K⁺ dependent dephosphorylation. In the presence of an inward K^? gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H⁺ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K⁺ conductance but a measurable H⁺ conductance, hence a K⁺ gradient can produce an H⁺ gradient in the presence of valinomycin. The uptake and spontaneous leak of H⁺ are temperature sensitive skin with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H⁺ uptake by these vesicles is probably due to a dimeric (H⁺ + K⁺)-ATPase and is probably non-electrogenic.     

Reconstitution and characterization of a sodium-stimulated active aminoisobutyric acid transport system derived from partially purified plasma membranes from mouse fibroblasts transformed by simian virus 40: comparison of reconstituted vesicles with native membrane vesicles

A Na⁺-specific and Na⁺-stimulated active α-aminoisobutyric acid transport system was reconstituted from plasma membranes isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40. The plasma membranes were treated with dimethylmaleic anhydride and then extracted with 2% cholate. The cholate-solubilized supernatant proteins were combined with exogenous phospholipids and eluted through a Sephadex G-50 column. This yielded reconstituted vesicles which in the presence of Na⁺ could actively transport α-aminoisobutyric acid as shown by the transient accumulation above the equilibrium level (overshoot). The overshoot was not obtained with other monovalent cations such as K⁺, Li⁺, and choline⁺. The electrochemical effect of the lipophilic anion, SCN^?, led to greater α-aminoisobutyric acid uptake as compared to that observed with Cl^? or SO₄^2?. The Na⁺-stimulated transport of a-aminoisobutyric acid was a saturable process with an apparent K_m of 2 mm. Studies of the inhibition of α-aminoisobutyric acid transport by other amino acids showed that methylaminoisobutyric acid [specifically transported by A system (alanine preferring)]had a pronounced inhibitory effect on a-aminoisobutyric acid uptake in contrast to the slight inhibitory effect produced by phenylalanine [primarily transported by L system (leucine preferring)]. The results show that the reconstituted vesicles, prepared from partially purified membrane proteins and exogenous phospholipids, regained the same important transport properties of native membrane vesicles, i.e., Na⁺-specific and Na⁺-stimulated concentrative α-aminoisobutyric acid uptake.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.