An ATP-driven proton pump in clathrin-coated vesicles

Clathrin containing coated vesicles prepared from bovine brain catalyzed ATP-driven proton translocation and a 32Pi-ATP exchange reaction. Both activities were measured in the presence of 5 micrograms of oligomycin/mg of protein which completely inhibited these reactions catalyzed by submitochondrial particles. Analyses performed during the purification procedure demonstrated that the oligomycin-resistant pump was concentrated and highly purified in the fractions containing coated vesicles. Moreover, vesicles precipitated by either monoclonal or polyclonal antibodies against clathrin contained the H+ pump activity. Dicyclohexylcarbodiimide (0.5 mM) and N-ethylmaleimide (1 mM) added to the assay mixture inhibited the pump completely, whereas neither vanadate, sodium azide, efrapeptin, or mitochondrial ATPase inhibitor had an effect.     

An ATP-driven proton pump in brush-border membranes from rat renal cortex

Summary The rate of ATP hydrolysis in ATP-preloaded plasma membrane vesicles derived from the luminal membrane of renal cortical tubules, and the rate of H⁺ secretion out of the same vesicles were investigated. Both were inhibited at low temperature, by the action of filipin, an antibiotic that complexes with cholesterol in plasma membranes, and by the action of blockers of mitochondrial F_o hydrogen channels, dicyclohexylcarbodiimide and Dio-9. Valinomycin in the presence of K⁺ showed a stimulatory effect, the protonophor carbonyl-cyanid-p-trifluormethoxy-phenylhydrazone stimulated the intravesicular ATP hydrolysis and apparently abolished acidification of the extravesccular medium. Lowering of the pH of the extravesicular medium retarded ATP hydrolysis, while readjustment of extra- and intravesicular pH accelerated ATP hydrolysis again. These findings strongly support the assumption that an ATP-driven proton pump is located in the luminal membrane of renal cortical tubules.     

ATP-driven,Na(+)-independent inward Cl- pumping in neuroblastoma cells

In immature neurones, the steady-state intracellular Cl- concentration [Cl-](i) is generally higher than expected for passive distribution, and this is believed to be due to Na(+)-K(+)-2Cl(-) co-transport. Here, we show that N2a neuroblastoma cells, incubated in HEPES-buffered NaCl medium maintain a [Cl-](i) around 60 mm, two- to threefold higher than expected for passive distribution at a membrane potential of - 49 mV. When the cells were transferred to a Cl(-) -free medium, [Cl-](i) decreased quickly (t(1/2) < 5 min), suggesting a high Cl- permeability. When the intracellular ATP concentration was reduced to less than 1 mm by metabolic inhibitors, the initial rate of (36) Cl- uptake was strongly inhibited (60-65%) while steady-state [Cl-](i) decreased to 24 mm, close to the value predicted from the Nernst equilibrium. Moreover, after reduction of [ATP](i) and [Cl-](i) by rotenone, the subsequent addition of glucose led to a reaccumulation of Cl-, in parallel with ATP recovery. Internal bicarbonate did not affect Cl- pumping, suggesting that Cl-/HCO(3)(-) exchange does not significantly contribute to active transport. Likewise, Na(+) -K(+) -2Cl(-) co-transport also appeared to play a minor role: although mRNA for the NKCC1 form of the co-transporter was detected in N2a cells, neither the initial rate of (36)Cl- uptake nor steady-state [Cl-](i) were appreciably decreased by 10 microm bumetanide or replacement of external Na(+) by choline. These results suggest that a highly active ATP-dependent mechanism, distinct from Na(+) -K(+) -2Cl(-) co-transport, is responsible for most of the inward Cl- pumping in N2a cells.     

Electrogenic Cl- pump in Acetabularia

Measurements of this transmembrane potential difference (V) under various conditions have demonstrated the operation of an electrogenic Cl- pump in the outer plasma membrane (plasmalemma) of the unicellular marine alga Acetabularia. In preparations of partly purified membranes (containing plasmalemma), there is Cl- stimulated, N,N'-dicyclohexylcarbodiimide-insensitive, vanadate-sensitive ATPase activity with a pH optimum around pH 6.5. These properties are consistent with the assumption that the electrogenic Cl- pump is an ATPase. In order to investigate electrical details of the "Mitchellian" type of charge-translocating enzyme, steady-state current-voltage curves of the electrogenic pump (Ip(V)) were measured in vivo under dark and light conditions and analysed by two-state reaction kinetic model. This model with the resulting parameters predicts V-sensitive, undirectional Cl- effluxes through the pump. The predictions of this model agree with the experimental results. Green light causes a fast decrease of V, which is explained as a disturbance of the pump cycle. Relaxation studies on this effect and reaction kinetic analysis of Ip(V) under different external Cl- concentrations are used to develop a consistent three-state model of the pump that includes the order of and absolute rate constants of individual reactions, states of charge, stoichiometry, voltage-sensitivity and density of the pump molecules in the membrane.     

Hepatic endosome fractions contain an ATP-driven proton pump.

Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.     

Evidence for an ATP-driven "proton pump" in rat liver lysosomes by basic dyes uptake.

When rat liver lysosomes are suspended in a medium containing acridine orange at neutral pH, accumulation of the dye may be observed within the vesicles. The uptake appears driven by a pH gradient between the external medium and the interior of the lysosomes since it is inhibited by NH₄⁺, nigericin and other electroneutral proton-cation exchangers. FCCP is ineffective in inhibiting the uptake. In the presence of Mg⁺⁺ and anions such as Cl^?, ATP promoted a further and more extensive but slower oligomycin and ouabain-insensitive dye uptake, which was also inhibited by FCCP. Very similar results were obtained with neutral red and atebrin. When the rate of the ATP-induced acridine uptake in preparations of different purification grade was compared, it was observed that the uptake rate increased in parallel with lysosomal enzymatic activity. These results suggest that an electrogenic ATP-driven-Mg⁺⁺ dependent “proton pump” is operating in the lysosomal membrane, as previously proposed.     

Stimulation of ATP-driven Ca2+ pump in the basal-lateral plasma membranes of kidney cortex during compensatory renal growth

During compensatory renal growth 45Ca2+ transport in basal-lateral plasma membrane vesicles isolated from the rat renal cortex have been investigated. Stimulation of Ca2(+)-ATPase activity was observed, without an effect of compensatory renal growth on Na+/Ca2+ exchanger activity and on passive Ca2+ permeability of the vesicles. Twelve hours following unilateral nephrectomy about 40% increase of Ca2(+)-ATPase activity above control value was observed and this effect was present until the end of the experimental period (7 days). When kinetic parameters for Ca2(+)-ATPase were studied in native membranes, an increase of Vmax was observed, whereas the Km for Ca2+ was similar in control vesicles and vesicles isolated from the remnant kidney. Depletion of endogenous calmodulin resulted in a decrease of Vmax and an increase of Km (Ca2+), while its addition reversed these parameters and increased the Hill coefficient from about 1 to about 2. Once again, only a significant increase of Vmax in vesicles isolated from the remnant kidney above the control value was observed. Finally, increase of Ca2(+)-ATPase activity during compensatory renal growth could be abolished by actinomycin D, indicating that its stimulation is due to protein synthesis.     

Aging reduces the GABA-dependent 36Cl- flux in rat brain membrane vesicles

The function of the chloride channel associated to GABAA receptor complex was analyzed in the brain of aged rats by measuring the chloride flux across the neuronal membrane and its modulation by drugs acting at the level of the GABA receptor complex and 35S-TBPS binding. The basal 36Cl- uptake by brain membrane vesicles of aged rats was higher (22%) than that observed in those of adult rats. The higher 36Cl- uptake found in cortical membrane vesicles of senescent rats was not sensitive to the action of bicuculline indicating that it was not the consequence of a tonic GABAergic modulation. Moreover, the stimulation of 36Cl- uptake induced by GABA was markedly lower in membrane vesicles of aged rats than that observed in those of adult rats. Accordingly, the stimulation of 36Cl- efflux elicited by GABA (18%) and pentobarbital (26%) was higher in membrane vesicles of adult rats with respect to that (8 and 16%, respectively) of old rats. Finally, a significant decrease of 35S-TBPS binding was observed in membrane preparation from the cerebral cortex, cerebellum and hippocampus of aged-rats. Scatchard plot analysis indicated that the decrease was entirely due to a reduction in the total number of binding sites with no change in their affinity. All together the results indicate that in the rat brain the function of the chloride channel coupled to the GABA/benzodiazepine/barbiturate receptor complex is reduced by aging.     

ATP-dependent Cl- uptake by plasma membrane vesicles from the rat brain

Uptake of Cl- by plasma membrane vesicles from the rat brain was stimulated by ATP at 37 degrees C, but not by beta, gamma-methylene ATP or at 0 degrees C. The addition of Triton X-100 or sucrose to the incubation medium diminished the ATP-stimulated Cl- uptake, suggesting that Cl- was transported across the membranes into the intravesicular space. This ATP-stimulated Cl- uptake was not affected by 1 mM ouabain. 1 microM oligomycin, 0.1 mM gamma-aminobutyric acid or 0.1 mM picrotoxin. Thus, non-mitochondrial ATP-driven Cl- transport through a system other than Na, K-ATPase or Cl- channels occurs in neuronal plasma membrane vesicles.     

ATP-driven Ca2+ pump in the basolateral membrane of rat kidney cortex catalyzes an electroneutral Ca2+/H+ antiport

An ATP-driven Ca2+ pump in the basolateral membrane of rat kidney cortex pumps Ca2+ out of the cell at the expense of MgATP (Km = 0.191 mM). This pump has a high affinity for free Ca2+ (26 nM). Vanadate, lanthanum, N-ethylmaleimide and calmodulin inhibitor R24571 inhibited this pump activity. Dimethyl[2-14C]oxazolidine-2,4-dione [( 14C]DMO) was entrapped in the vesicles in association with the ATP-driven Ca2+ influx. The ATP-driven Ca2+ influx was stimulated by the intravesicular acid pH and an upper convex Lineweaver-Burk reciprocal plot suggested two possible kinetics; one is that this Ca2+ pump is an allosteric enzyme with more than 1.72 H+ binding sites and another is the presence of two Ca2+ pumps with different affinities for H+. Valinomycin study indicated that the ATP-dependent Ca2+ transport by the BLMV was electroneutral and voltage independent. These results strongly suggest that the ATP-driven Ca2+ pump in the renal basolateral membrane catalyzes an electroneutral Ca2+/H+ antiport.     

ATP-driven succinate oxidation in the catabolism of Desulfuromonas acetoxidans

The oxidation of succinate with elemental sulphur in Desulfuromonas acetoxidans was investigated using a membrane preparation of this bacterium. The following results were obtained:

1. The preparation catalyzed the oxidation of succinate with sulphur and NAD. These reactions were dependent on ATP and were abolished by the presence of protonophores or dicyclohexylcarbodiimide (DCCD).
2. The membrane preparation also catalyzed the reduction of fumarate with H₂S or with NADH. These activities were not dependent on ATP and were not affected by protonophores or DCCD.
3. By extraction-reincorporation experiments it could be shown that menaquinone is involved in electron transport between H₂S and fumarate and between NADH and fumarate.
4. The membrane fraction catalyzed the reduction of the water-soluble menaquinone-analogue dimethylnaphthoquinone (DMN) by succinate, H₂S, or NADH, and the oxidation of DMNH₂ by fumarate. These activities were not dependent on the presence of menaquinone and were not influenced by ATP.
5. The activities involving succinate oxidation or fumarate reduction were similarly sensitive to 2(n-nonyl)-4-hydroxyquinoline-N-oxide, while H₂S and NADH oxidation by DMN were not affected by the inhibitor.

It is concluded that the catabolism of D. acetoxidans involves the energy-driven oxidation of succinate with elemental sulphur or NAD as electron acceptors and that menaquinone is a component of the electron transport chain catalyzing these reactions.     

The branchial chloride pump in the goldfish Carassius auratus: relationship between Cl-/HCO3- and Cl-/Cl-- exchanges and the effect of thiocyanate.

1. The effect of thiocyanate on chloride and sodium fluxes across the gill was studied in the goldfish Carassius auratus. At low external chloride concentrations, addition of SCN- to the bathing solution markedly inhibited chloride influx and efflux, the net flux being reversed, SCN- injection was without effect. SCN- had no effect on sodium fluxes when injected or added to the external medium. 2. The inhibition of chloride influx by SCN- was of a mixed type involving simultaneous modifications of the affinity constant of the carrier for Cl- and of the maximal Cl- influx. The affinity constant of the carrier for SCN- was 10 times lower than that for Cl-. 3. The gill of the goldfish was found to be practically impermeable to SCN-. 4. In the presence of external SCN-, the Cl-/HCO3- exchange was reversed: Cl- was lost against HCO3- which is absorbed. This suggests an obligatory exchange. 5. Exchange diffusion for chloride was also demonstrated. 6. A kinetic model is proposed to explain chloride and bicarbonate transport across the gill of Carassius auratus.     

The Aplysia californica Cl- pump is a P-type ATPase: evidence through inhibition studies

Utilizing a proteoliposomal preparation containing Cl(-)-ATPase from Aplysia californica foregut, it was shown that orthovanodate inhibited Cl(-)-ATPase activity, ATP-dependent Cl- transport, ATP-dependent membrane potential change and ATP-dependent phosphorylation. N-ethylmalemide and p-chloromercurobenzoate also inhibited the Cl- pump biochemical and physiological transport characteristics. However, bafilomycin, azide, N, N'-dicyclohexylcarboiimide (DCCD), and efrapeptin had no effect on the Cl- pump biochemical or physiological characteristics, suggesting that this Cl- pump was a P-type ATPase. It was concluded that this P-type ATPase Cl- pump is the mechanism that is responsible for the net absorptive flux of Cl- in the A. californica foregut.     

ATP-driven electron transfer in enzymatic radical reactions

A novel method to generate organic radicals in enzymatic reactions is described, which is similar to electron transfer in nitrogenase. Component A of 2-hydroxyglutaryl-CoA dehydratase contains a [4Fe-4S] cluster located at the interface between its two identical subunits. The cluster is reduced by one electron derived from ferredoxin or flavodoxin. Hydrolysis of two ATP bound to component A, one to each subunit, enhances the reductive power of the electron and transfers it to component D, the actual dehydratase, where a low potential [4Fe-4S](2+) cluster is probably reduced. Further transfer to the substrate (R)-2-hydroxyglutaryl-CoA probably generates a substrate-derived ketyl radical anion, which expels the adjacent hydroxyl group. The resulting enoxy radical is deprotonated to a product-related ketyl radical anion. Finally the electron is removed by the next incoming substrate leading to the product glutaconyl-CoA and starting a new turnover. A similar, but stoichiometric rather than catalytic electron transfer has been established for the related benzoyl-CoA reductase.     

Osmotic regulation of the sodium pump in rat brain synaptosomes]

Effect of medium osmolarity on 3H-ouabain binding and the rate of ouabain-sensitive 86Rb+ transport in the rat brain synaptosomes was studied. A decrease in tonicity to 230 mOsm increases both parameters indicating the activation of the sodium pump upon synaptosome swelling. The effect is retained in the absence of inside-oriented Na+ gradient, i. e. a rise in Na(in) is not responsible for hypoosmotic activation. Colchicine (5mM) decreased and cytochalasin B (40 microM) increased the ouabain binding. In the presence of cytochalasin B the inhibition of binding observed under hypotonic conditions was shifted to higher osmolarity values. It is suggested that volume regulation of the sodium pump is controlled by the cytoskeleton elements.     

Properties of ATP-driven reverse electron flow in chloroplasts.

1. The reverse reactions induced by coupled ATP hydrolysis were studied in spinach chloroplasts by measurements of the ATP-induced increase in chlorophyll fluorescence reflecting reverse electron flow, and of the ATP-induced decrease in 9-aminoacridine fluorescence, representing formation of the transthylakoidal proton gradient (deltapH). ATP-induced reverse electron flow was kinetically analysed into three phases, of which only the second and third one were paralleled by corresponding phases in deltapH formation. The rapid first phase and formation of a deltapH occur also in the absence of the electron transfer mediator phenazine methosulfate. 2. The rate and extent of the reverse reactions were measured at temperatures in the range from 0 to 30 degrees C. The rate of formation of delta pH and of reverse electron flow were faster at high temperatures, but the maximal extent of delta pH and chlorophyll fluorescence increase were observed at the lowest temperature. Considering rate and extent of the ATP-stimulated reactions, a temperature optimum around 15 degrees C was found. Light activation of the ATPase occurred throughout the range studied. At 0 degrees C and in the presence of inorganic phosphate the activated state for ATPase was maintained for more than 10 min. 3. The ATP-induced rise in chlorophyll fluorescence yield was found to be of similar magnitude as the rise induced by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU), when both were measured with an extremely weak measuring beam. It is concluded, that both effects, although derived via distinctly different pathways, are limited by the same electron donating or electron accepting pool.     

Properties of ATP-driven reverse electron flow in chloroplasts

1. The reverse reactions induced by coupled ATP hydrolysis were studied in spinach chloroplasts by measurements of the ATP-induced increase in chlorophyll fluorescence reflecting reverse electron flow, and of the ATP-induced decrease in 9-aminoacridine fluorescence, representing formation of the transthylakoidal proton gradient (ΔpH). ATP-driven reverse electron flow was kinetically analysed into three phases, of which only the second and third one were paralleled by corresponding phases in ΔpH formation. The rapid first phase and formation of a ΔpH occur also in the absence of the electron transfer mediator phenazine methosulfate.2. The rate and extent of the reverse reactions were measured at temperatures in the range from 0 to 30°C. The rate of formation of ΔpH and of reverse electron flow were faster at high temperatures, but the maximal extent of ΔpH and chlorophyll fluorescence increase were observed at the lowest temperature. Considering rate and extent of the ATP-stimulated reactions, a temperature optimum around 15°C was found. Light activation of the ATPase occurred throughout the range studied. At 0°C and in the presence of inorganic phosphate the activated state for ATPase was maintained for more then 10 min.3. The ATP-induced rise in chlorophyll fluorescence yield was found to be of similar magnitude as the rise induced by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), when both were measured with an extremely weak measuring beam. It is concluded, that both effects, although derived via distinctly different pathways, are limited by the same electron donating or electron accepting pool.