Energy transduction in Escherichia coli. The effect of chaotropic agents on energy coupling in everted membrane vesicles from aerobic and anaerobic cultures.

1. The transduction of energy from the oxidation of substrates by the electron transport chain or from the hydrolysis of ATP by the Mg2+-ATPase was measured in everted membrane vesicles of Escherichia coli using the energy-dependent quenching of quinacrine fluorescence and the active transport of calcium. 2. Treatment of everted membranes derived from a wild-type strain with the chaotropic agents guanidine-HC1 and urea caused a loss of energy-linked functions and an increase in the permeability of the membrane to protons, as measured by the loss of respiratory-linked proton uptake. 3. The coupling of energy to the quenching of quinacrine fluorescence and calcium transport could be restored by treatment of the membranes with N,N'-dicyclohyexylcarbodiimide. 4. Chaotrope-treated membranes were found to lack Mg2+-ATPase activity. Binding of crude soluble Mg2+-ATPase to treated membranes restored energy-linked functions. 5. Membranes prepared from a wild-type strain grown under anaerobic conditions in the presence of nitrate retained respiration-linked quenching of quinacrine fluorescence and active transport of calcium after treatment with chaotropic agents. 6. Everted membrane vesicles prepared from an Mg2+-ATPase deficient strain lacked respiratory-driven functions when the cells were grown aerobically but were not distinguishable from membranes of the wild-type when both were grown under anaerobic conditions in the presence of nitrate. 7. It is concluded (a) that chaotropic agents solubilize a portion of the Mg2+-ATPase, causing an increase in the permeability of the membrane to protons and (b) that growth under anaerobic conditions in the presence of nitrate prevents the increase in proton permeability caused by genetic or chemical removal of the catalytic portion of the Mg2+-ATPase.     

Leishmania plasma membrane Mg2+-ATPase is a H+/K+-antiporter involved in glucose symport. Studies with sealed ghosts and vesicles of opposite polarity

Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg2+ (1 mm). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K+ (20 mm) that was inhibitable (>85%) with SCH28080 (50 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+(1:1) was established. Rapid and 10-fold active accumulation of [U-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H+/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.     

Adenosine 5''-triphosphate synthesis energized by an artificially imposed membrane potential in membrane vesicles of Escherichia coli.

Adenosine 5'-triphosphate (ATP) synthesis driven by an artificially imposed membrane potential in right-side-out membrane vesicles of Escherichia coli was investigated. Membrane vesicles prepared in the presence of adenosine diphosphate were loaded with K+ by incubation with 0.5 M potassium phosphate. Addition of valinomycin resulted in the synthesis of 0.2 to 0.3 nmol of ATP/mg of membrane protein, whereas no synthesis was observed after addition of nigericin. Addition of K+, dicyclohexylcarbodiimide, carbonylcyanide p-trifluoromethoxyphenylhydrazone, or azide to the assay buffer inhibited ATP synthesis. Adenosine diphosphate and Mg2+ were found to be required. Ca2+, which can replace Mg2+ for the hydrolytic activity of the Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3), could not replace Mg2+ in the synthetic reaction and, in fact, inhibited ATP synthesis even in the presence of Mg2+. Strain NR-70, a mutant lacking the Mg2+-ATPase, was unable to synthesize ATP using an artificially imposed membrane potential. Additionally, the Mg2+-ATPase was found to contain tightly bound ATP.     

Characterization of the ATP-dependent proton pump of clathrin-coated vesicles

The ATP-dependent proton pump which was previously identified in clathrin-coated vesicles isolated from calf brain (Forgac, M., Cantley, L., Wiedenmann, B., Altstiel, L., and Branton, D. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1300-1303) is further characterized. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) was identified as a potent inhibitor of both ATP-dependent proton uptake and Mg2+-ATPase activity of coated vesicles. Thus, incubation with 10 microM NBD-Cl for 10 min at 23 degrees caused the loss of 80% of the Mg2+-ATPase activity and 95% of the proton pumping activity. The observed protection from NBD-Cl inhibition by ATP suggests that NBD-Cl may react at the catalytic site, and reversal of NBD-Cl inhibition by 2-mercaptoethanol is consistent with reaction at either a tyrosine or cysteine residue. In addition, no stable phosphorylated intermediate was observed during turnover of the coated vesicle proton pump and neither Na+ nor K+ was countertransported by the pump during ATP-dependent proton uptake.     

Studies on the Proton Pumping Activity of H+-ATPase in Tonoplast Vesicles of Populus euphratica

Tonoplast-enriched vesicles were prepared from suspension-cultured Populus euphratica Oliv. cells by differential centrifugation and discontinuous sucrose density gradient centrifugation. The properties of the proton pumping activity of H+-ATPases in tonoplast vesicles were studied by acridine orange fluorescent quenching measured at 22 ℃. The proton pumping activity of ATPase was ATP-dependent with apparent Michaelis-Menten Constant (Km) for ATP about 0.65 mmol/L. The optimal pH for H+-ATPases activity was 7.5. The proton pumping activity of H+-ATPase could be initiated by some divalent cations, Mg2+ being highly efficient, much more than Fe2+; and Ca2+, Cu2+ and Zn2+ were inefficient under the experimental condition. The proton translocation could be stimulated by halide anions, with potencies decreasing in the order Cl-> Br->I->F-. The proton pumping activity was greatly inhibited by N-ethylmaleimide (NEM), N,N′-dicyclohexylcarbodiimide (DCCD), NO-3 and Bafilomycin A1, but not by orthovanadate and azide. These results demonstrated that the H+-ATPase in the tonoplast of Populus euphratica belonged to vacuolar type ATPase. This work was the first time that tonoplast-enriched vesicles were isolated from Populus euphratica cells.     

Characterization of Calcium-Activated and Magnesium-Activated ATPases of Brain Nerve Endings

The properties of Ca2+-activated and Mg2+-activated ATPases of nerve endings from mouse brain were investigated. Ca2+ and Mg2+ each can activate ATP hydrolysis in synaptosomes and its subfractions. Both Ca2+-ATPase and Mg2+-ATPase exhibit high and low affinity for their respective cations. At millimolar concentrations of Ca2+ or Mg2+, several nucleoside triphosphates could serve as substrate for the two enzymes and their specific activities were about three to four times higher in synaptic vesicles than in synaptosomal plasma membranes (SPM). Both in SPM and in synaptic vesicles the relative activity in the presence of Ca2+ was in the order of CTP greater than UTP greater than GTP = ATP, but with Mg2+ the activity was higher with ATP than with the other three triphosphates. Mg2+-ATPase was more active than Ca2+-ATPase in SPM, but in synaptic vesicles the two enzymes exhibited similar activity. Kinetic studies revealed that Mg2+-ATPase was inhibited by excess ATP and not by excess Mg2+. The simultaneous presence of Na+ + K+ stimulated Mg2+-ATPase and inhibited Ca2+-ATPase activity in intact synaptosomes and SPM. The stimulation of Mg2+-ATPase by Na+ + K+ was further increased by increasing Mg2+ concentration and was inhibited by Ca2+ and by ouabain. When Ca2+ and Mg2+ are present together in SPM or synaptic vesicles, the total Pi liberated by the two cations may either increase or decrease, depending on their relative concentrations. Kinetic analyses indicate that Ca2+ and Mg2+ bind independently to the enzyme alone or together at different sites. The results suggest that Ca2+-ATPase and Mg2+-ATPase in SPM or synaptic vesicles may be separate and distinct systems.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

Boar acrosin is a two-chain molecule. Isolation and primary structure of the light chain; homology with the pro-part of other serine proteinases

Cholinergic synaptic vesicles from the electric organ of Torpedo marmorata are associated with a Mg2+-ATPase insensitive to ouabain and oligomycin. Treatment of vesicle membranes with dichloromethane releases a Mg2+-ATPase with apparent molecular mass of around 250 kDa as determined by gel filtration. The vesicular ATPase resembles the mitochondrial F1-ATPase in these properties. Gel electrophoresis of the solubilized ATPase shows however that only a single 50-kDa band is present as compared to the alpha-subunit (52 kDa) and beta-subunit (50 kDa) of electric organ mitochondrial F1-ATPase present in this range of molecular mass range. In agreement, covalent photoaffinity labelling of isolated vesicles with azido-ATP shows a 50-kDa band. Vesicle ghosts were found to accumulate [14C]methylamine in an ATP-dependent manner indicating the presence of an inwardly directed proton pump. We conclude that cholinergic vesicles contain a proton pump probably driven by the Mg2+-ATPase here described, which generates an electrochemical gradient across the vesicle membrane and is necessary for uptake and storage of acetylcholine within the vesicles.     

Calcium transport driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae

Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulate Ca2+ ion in the presence of ATP, not in the presence of ADP or adenyl-5'-yl imidodiphosphate. Calcium transport showed saturation kinetics with a Km value of 0.1 mM and optimal pH of 6.4. Ca2+ ion incorporated in the vesicles was exchangeable and released completely by a protonophore uncoupler, 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847), or calcium-specific ionophore, A23187. The transport required Mg2+ ion but was inhibited by Cu2+ or Zn2+ ions, inhibitors of H+-ATPase of the vacuolar membrane. The transport activity was sensitive to the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to oligomycin or sodium vanadate. SF6847 or nigericin blocked Ca2+ uptake completely, but valinomycin stimulated it 1.35-fold. These results indicate that an electrochemical potential difference of protons is a driving force for this Ca2+ transport. The ATP-dependent formation of the deltapH in the vesicles and its partial dissipation by CaCl2 were demonstrated by fluorescence quenching of quinacrine. This Ca2+ uptake by vacuolar membrane vesicles is suggested to be catalyzed by a Ca2+/H+ antiport system.     

Calmodulin regulation of Ca2+ transport in human erythrocytes.

Inside-out vesicles of human erythrocytes took up Ca2+ against an electrochemical gradient. This Ca2+ uptake was dependent on ATP and was stimulated by calmodulin. Treatment of vesicles with 1 mM-EDTA exposed an apparent low-CA2+-affinity Ca2+-transport component with Kd of about 100 microM-Ca2+ or more. This was converted into a single high-Ca2+-affinity transport activity of Kd about 2.5 microM-Ca2+ in the presence of 2 micrograms of calmodulin/ml, showing that the decrease in transport activity after EDTA treatment was reversible. Vesicles not extracted with EDTA showed mainly apparent high-Ca2+-affinity kinetics even in the absence of added calmodulin. Trifluoperazine (30 microM) and calmodulin-binding protein (20 micrograms/ml) inhibited about 50% of the high-affinity Ca2+ uptake and (Ca2+ + Mg2+)-ATPase (Ca2+-activated, Mg2+-dependent ATPase) activity of these vesicles, indicating that the vesicles isolated by the procedure used retained some calmodulin from the erythrocytes. Comparison of Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities in inside-out vesicles yielded a variable Ca2+/P1 stoichiometric ratio. At low free Ca2+ concentrations (below 20 micro-Ca2+), a Ca2+/P1 ration of about 2 was found, whereas at higher Ca2+ concentrations the stoichiometry was approx. 1. The stoichiometry was not significantly altered by calmodulin.     

Purification and characterization of the F1 ATPase from Bacillus subtilis and its uncoupler-resistant mutant derivatives.

The F1 ATPase of Bacillus subtilis BD99 was extracted from everted membrane vesicles by low-ionic-strength treatment and purified by DEAE-cellulose chromatography, hydrophobic interaction chromatography, and anion-exchange high-performance liquid chromatography. The subunit structure of the enzyme was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of urea. In the absence of urea, the alpha and beta subunits comigrated and the ATPase was resolved into four bands. The mobility of the beta subunit, identified by immunoblotting with anti-beta from Escherichia coli F1, was altered dramatically by the presence of urea, causing it to migrate more slowly than the alpha subunit. The catalytic activity of the ATPase was strongly metal dependent; in the absence of effectors, the Ca2+-ATPase activity was 15- to 20-fold higher than the Mg2+ -ATPase activity. On the other hand, sulfite anion, methanol, and optimally, octylglucoside stimulated the Mg2+ -ATPase activity up to twice the level of Ca2+ -ATPase activity (specific activity, about 80 mumol of Pi per min per mg of protein). The F1 ATPase was also isolated from mutants of B. subtilis that had been isolated and characterized in this laboratory by their ability to grow in the presence of protonophores. The specific activities of the ATPase preparations from the mutant and the wild type were very similar for both Mg2+- and Ca2+ -dependent activities. Kinetic parameters (Vmax and Km for Mg-ATP) for octylglucoside-stimulated Mg2+ -ATPase activity were similar in both preparations. Structural analysis by polyacrylamide gel electrophoresis and isoelectric focusing indicated that the five F1 subunits from ATPase preparations from the mutant and wild-type strains had identical apparent molecular weights and that no charge differences were detectable in the alpha and beta subunits in the two preparations. Thus, the increased ATPase activity that had been observed in the uncoupler-resistant mutants is probably not due to a mutation in the F1 moiety of the ATPase complex.     

Functional mosaicism of membrane proteins in vesicles of Escherichia coli.

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.     

Properties of H+-translocating adenosine triphosphatase in vacuolar membranes of SAccharomyces cerevisiae

The properties of Mg2+-ATPase in the vacuole of Saccharomyces cerevisiae were studied, using purified intact vacuoles and right-side-out vacuolar membrane vesicles prepared by the method of Y. Ohsumi and Y. Anraku ((1981) J. Biol. Chem. 256, 2079). The enzyme requires Mg2+ ion but not Ca2+ in. Cu2+ and Zn2+ ions inhibit the activity. The optimal pH is at pH 7.0. The enzyme hydrolyzes ATP, GTP, UTP, and CTP in this order and the Km value for ATP was determined as 0.2 mM. It does not hydrolyze ADP, adenosyl-5'-yl imidodiphosphate, or p-nitrophenyl phosphate. ADP does not inhibit hydrolysis of ATP by the enzyme. The activities of intact vacuoles and of vacuolar membrane vesicles were stimulated 3- and 1.5-fold, respectively, by the protonophore uncoupler 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile and the K+/H+ antiporter ionophore nigericin. Sodium azide at a concentration exerting an uncoupler effect also stimulated the activity. The activity was sensitive to the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to sodium vanadate. The ATP-dependent formation of an electrochemical potential difference of protons, measured by the flow-dialysis method, was determined as 180 mV, with contribution of 1.7 pH units, interior acid, and of a membrane potential of 75 mV. It is concluded that the Mg2+-ATPase of vacuoles is a new marker enzyme for these organelles and is a N,N'-dicyclohexylcarbodiimide-sensitive, H+-translocating ATPase whose catalytic site is exposed to the cytoplasm.     

Proton pump-linked Mg2+-ATPase activity in isolated rat liver lysosomes

ATPase activity in highly purified rat liver lysosome preparations was evaluated in the presence of other membrane cellular ATPase inhibitors, and compared with lysosome ATP-driven proton translocating activity. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 50% inhibition in divalent cation-dependent ATPase activity, and an 80% inactivation of ATP-linked lysosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+, ATPase activity was similar to that seen in an Mg2+ medium. Mg2+-dependent ATPase activity was greatly inhibited (from 70 to 80%) by the platinum complexes; cis-didimethylsulfoxide dichloroplatinum(II) (CDDP) at approximately 90 microM and cis-diaminedichloroplatinum(II) at twofold higher concentrations. Less inhibition, about 30 and 45%, was obtained with N,N'-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. The concentration dependence of inhibition by the above drugs was determined for both proton pumping and ATPase activities, and half-maximal inhibition concentration of each activity was found at nearly similar values. A micromolar concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented ATP from setting up a pH gradient across the lysosomal membranes, but stimulated Mg2+-ATPase activity significantly. ATPase activity in Ca2+ medium was also inhibited by CDDP and stimulated by FCCP, but both effects were two- to threefold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in a CDDP-supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton pump, was anion dependent. The lowest activity was recorded in a F-medium, and increased in the order of F- less than SO2-4 less than Cl- approximately equal to Br-. The CDDP-sensitive ATPase activity observed, supported by Mg2+ and less so by Ca2+, may be related to lysosome proton pump activity.     

Association of (Ca+Mg)-ATPase activity with ATP-dependent Ca uptake in vesicles prepared from human erythrocytes

Ghost membranes prepared from human erythrocytes exhibit 2 distinct (Ca+Mg)-ATPase¹ activities (Quist and Roufogalis, Arch Biochem Biophys 168:240, 1975). (Ca+Mg)-ATPase activity dependent on a water soluble protein fraction is selectively lost from ghost membranes during preparation of vesicles under low ionic strength, slightly alkaline conditions. In this study, the Ca²⁺ dependence of the remaining membrane bound (Ca+Mg)-ATPase activity and ATP-dependent Ca uptake in vesicles were compared. The C²⁺ activation curves for (Ca+Mg)-ATPase activity and Ca uptake into vesicles were parallel over a Ca²⁺ range of 0.3–330 μM, and both curves have 2 apparent K_A values for Ca²⁺ of 0.45 and 100 μM. Addition of a concentrated soluble protein fraction containing predomintly spectrin to the vesicles increased (Ca+Mg)-ATPase activity over twofold but did not affect the rate of Ca uptake. These findings suggest that the (Ca+Mg)-ATPase activity remaining in vesicles after extraction of the water soluble proteins is associated with the Ca pump whereas (Ca+Mg)-ATPase activity dependent on the soluble protein fraction is associated with some other function.     

The involvement of altered vesicle transport in redistribution of Ca2+, Mg2+-ATPase in cholestatic rat liver

Vectorial sorting of plasma membrane protein-containing vesicles is essential for the establishment and maintenance of cell polarity. In the present study, the involvement of altered vesicle transport in the redistribution of membrane-bound Ca2+, Mg2+-ATPase resulting from cholestasis was investigated in hepatocytes. Cholestasis was induced in rat liver by common bile duct ligation. Ca2+, Mg2+-ATPase activity was demonstrated histochemically at the light and electron microscopical levels. Microtubules, an important factor for transcellular transport of vesicles, were studied in situ by immunofluorescence microscopy and electron microscopy in detergent-extracted preparations. The results showed that microtubules underwent significant changes after common bile duct ligation. The most pronounced alteration was focal accumulation of -tubulin in the cytoplasm of hepato cytes after 7 days of common bile duct ligation. At the electron microscopical level, the number of microtubules was increased considerably. In control livers, the activity of Ca2+, Mg2+-ATPase was localized only at the apical plasma membrane of hepatocytes, but it was also present at the basolateral plasma membrane after common bile duct ligation. The number of intracellular vesicles containing Ca2+, Mg2+-ATPase activity was increased strikingly, and some of them were associated with lateral membrane domains in which Ca2+, Mg2+-ATPase activity was found. It is concluded that common bile duct ligation induces the rearrangement of microtubules, which may disturb vectorial transport of Ca2+, Mg2+-ATPase-containing vesicles in hepatocytes, leading to the redistribution of Ca2+, Mg2+-ATPase. © 1998 Chapman & Hall     

Orientation of synaptic plasma membrane vesicles containing calcium pump and sodium-calcium exchange activities

Sidedness of synaptic plasma membrane vesicles isolated from brain synaptosomes has been assessed by two distinct experimental approaches: first, analysis of (Na+ + K+)-ATPase, Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities before and after permeabilization of vesicles; second, analysis of Ca2+ fluxes via the Na+/Ca2+ exchanger, before and after modification of an imposed Na+ gradient by penetrating or nonpenetrating Na+ channel-modifying drugs. 0.05% saponin, which completely permeabilizes the vesicles, increases digitoxigenin-sensitive (Na+ + K+)-ATPase, basal Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities by 51.0, 47.4, and 83.6%, respectively. Saponin increases only the Vmax of the latter activity, the Km for Ca2+ (0.13 microM; the same as that for Ca2+-pumping) being unaltered by saponin. An increment of 20.5% in the Vmax of (Ca2+ + Mg2+)-ATPase activity with 10 microM A23187, reveals that the enzyme activity in nonpermeabilized vesicles is limited by the formation of a Ca2+ gradient. Thus, the saponin-induced increment in (Ca2+ + Mg2+)-ATPase due only to exposure of occluded sites (as opposed to Ca2+ gradient dissipation) is actually 52%, which is similar to values for both other ATPases, and suggests that 32-35% of plasma membranes exist in an inverted orientation. Vesicle orientation was independently assessed by the differential actions of tetrodotoxin (a membrane impermeant blocker) and veratridine (a membrane permeant agonist) on Na+-channel opening measured indirectly by dissipation of an imposed Na+ gradient utilized to drive a large 45Ca2+ accumulation via the Na+/Ca2+ exchanger. Tetrodotoxin reverses 35-44% of veratridine-mediated Na+ gradient-dissipation, the relative membrane-permeability of the two channel modifiers, suggesting that 56-65% of sealed vesicles are inverted. The concurrence of these two independent measurements of vesicle orientation reinforces their validity.     

Plasma Membrane H+-ATPase Activity in Spores, Germ Tubes, and Haustoria of the Rust Fungus Uromyces viciae-fabae

Using plasma membrane-enriched vesicles, the properties of the H+-ATPase (EC 3.6.1.35) from the rust fungus Uromyces viciae-fabae were studied. The enzyme is strictly Mg2+-dependent and is inhibited by vanadate. The pH-optimum is at 6.7. By Western blot analysis using a monoclonal antibody against corn plasma membrane H+-ATPase a polypeptide of approximately 104 kDa could be detected. The vanadate-sensitive H+-ATPase activity of microsomal vesicles obtained from different stages of rust development was determined. Uredospores had only a very low enzyme activity (1.9 μmol Pi x mg-1 protein x h-1). In germ tubes the ATPase activity was about twofold higher (4.0 μmol Pi x mg-1 protein x h-1). An eightfold higher ATPase activity (16.1 μmol Pi x mg-1 protein x h-1) was found in microsomal vesicles from haustoria which had been isolated from rust-infected Vicia faba leaves. These results suggest, that the electrochemical gradient generated by the H+-ATPase of haustoria plays an important role for their function, possibly by promoting nutrient uptake from host cells.     

Uptake of γ-Aminobutyric Acid by a Synaptic Vesicle Fraction Isolated from Rat Brain

Gamma-Aminobutyric acid (GABA) was taken up by a MgATP-dependent mechanism into synaptic vesicles isolated by hypoosmotic shock and density gradient centrifugation. The properties of the vesicular uptake differed clearly from those of synaptosomal and glial uptake, both with respect to Na+, Mg2+, and ATP dependence and with respect to response to general GABA uptake inhibitors such as nipecotic acid, diaminobutyric acid, and beta-alanine. The uptake showed a Km of 5.6 mM and a net uptake rate of 1,500 pmol/min/mg of protein. It is suggested that the vesicular uptake of GABA is driven by an electrochemical proton gradient generated by a Mg2+-ATPase.     

Redistribution and characterization of (H+ + K+)-ATPase membranes from resting and stimulated gastric parietal cells.

When isolated from resting parietal cells, the majority of the (H+ + K+)-ATPase activity was recovered in the microsomal fraction. These microsomal vesicles demonstrated a low K+ permeability, such that the addition of valinomycin resulted in marked stimulation of (H+ + K+)-ATPase activity, and proton accumulation. When isolated from stimulated parietal cells, the (H+ + K+)-ATPase was redistributed to larger, denser vesicles: stimulation-associated (s.a.) vesicles. S.a. vesicles showed an increased K+ permeability, such that maximal (H+ + K+)-ATPase and proton accumulation activities were observed in low K+ concentrations and no enhancement of activities occurred on the addition of valinomycin. The change in subcellular distribution of (H+ + K+)-ATPase correlated with morphological changes observed with stimulation of parietal cells, the microsomes and s.a. vesicles derived from the intracellular tubulovesicles and the apical plasma membrane, respectively. Total (H+ + K+)-ATPase activity recoverable from stimulated gastric mucosa was 64% of that from resting tissue. Therefore, we tested for latent activity in s.a. vesicles. Permeabilization of s.a. vesicles with octyl glucoside increased (H+ + K+)-ATPase activity by greater than 2-fold. Latent (H+ + K+)-ATPase activity was resistant to highly tryptic conditions (which inactivated all activity in gastric microsomes). About 20% of the non-latent (H+ + K+)-ATPase activity was also resistant to trypsin digestion. We interpret these results as indicating that, of the s.a. vesicles, approx. 55% have a right-side-out orientation and are impermeable to ATP, 10% right-side-out and permeable to ATP, and 35% have an inside-out orientation.