Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.     

ATP-dependent proton translocation and quenching of 9-aminoacridine fluorescence in inside-out membrane vesicles of a cytochrome-deficient mutant of Escherichia coli,.

1. ATP-dependent proton translocation and ATP-dependent quenching of the fluorescence of 9-aminoacridine were measured in inside-out vesicles derived from a cytochrome-deficient mutant of Escherichia coli. 2. ATP-dependent quenching of fluorescence was inhibited by nigericin gramicidin, NH4Cl, and carbonylcyanide-m-chlorophenylhydrazone. Inhibition was also produced by the ATPase inhibitors N,N'-dicyclohexylcarbodimide (DCCD) and diphenyl phosphorazidate (DPA), and by the respiratory chain inhibitors piericidin A, 2-heptyl-4-hydroxyquinoline N-oxide, and An2+. The inhibition of ATP-dependent fluorescence quenching by the ionophores, uncouplers, and respiratory chain inhibitors was not due to an effect on ATPase activity which was insensitive to these agents. 3. By use of the ATPase inhibitors DCCD and DPA, or by replacing ATP with GTP, ITP and CTP, a correlation between the ATPase activity and the rate of ATP-dependent membrane energization, as measured by fluorescence quenching, was obtained.     

Characterization of a proton translocating ATPase and sucrose uptake in a tonoplast-enriched vesicle fraction from sugarcane

A sucrose gradient fraction was used to characterize the tonoplast ATPase from storage tissue of the sugarcane plant ( Saccharum sp. var. H57–5175). Marker enzyme analyses and characterization of low-density vesicles isolated on a sucrose gradient were consistent with a highly enriched tonoplast fraction. ATPase and proton transport activities were both substantially inhibited by nitrate (80%), but very little by vanadate (10%), indicating a high titer of tonoplast compared to plasma-membrane vesicles in the fraction. Sensitivity toward other inhibitors, as well as ion effects, correlated closely among ATPase and proton translocation activities. Although the vesicles in this fraction showed good proton translocating activity there was no indication that ATP stimulated sucrose uptake in this tonoplast population.     

Specificity of association of a Ca2+/Mg2+ ATPase with cholinergic synaptic vesicles from Torpedo electric organ.

Cholinergic synaptic vesicles from the electric organ of $\text{Torpedo}$$\text{californica}$ have been subjected to analytical scale separation techniques not utilized in the isolation procedure, and the ATPase activity of separated fractions determined. Most of the ATPase activity migrated with the vesicles. Sensitivity of the ATPase activity to 16 potential inhibitors also was determined. Most of the ATPase activity was inhibited by low concentrations of 4-chloro-7-nitrobenzo-oxadiazole (NBD-C1) and dicyclohexylcarbodiimide (DCCD), but not by a water soluble carbodiimide. The close association of the ATPase with the vesicles and the pattern of inhibition obtained provide further support for the authentic presence of a membrane bound $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase in the cholinergic synaptic vesicle.     

Stimulation of canine kidney BBMV ATPase activity by acidic pH in the presence of Zn2+: an ATPase activity distinct from transport ATPases and alkaline phosphatase that may be an ecto-ATPase

Renal brush border membrane vesicles (BBMV) of the dog possess at least two ATPase activities. In the present study, we have examined the effect of pH, ions, and inhibitors on the activity of ATPase in BBMV. Two different sets of conditions were identified that produced stimulation of ATPase activity. A unique stimulation of BBMV ATPase activity occurred at acidic pH in the presence of 1 mM ZnCl2. In the absence of Zn2+, a second ATPase activity was stimulated by alkaline pH values with peak stimulation occurring between pH 8.5 and 9.0. The results suggest that the alkaline pH-stimulated hydrolysis of ATP probably represents the activity of BBMV alkaline phosphatase. The unique acidic pH + Zn2(+)-stimulated ATPase activity must represent the activity of a second protein other than the alkaline phosphatase, since purified alkaline phosphatase did not show this activity. The biochemical identity and physiological function of this renal BBMV ATPase activity remain to be determined, but it may be an ecto-ATPase.     

Cholinergic Synaptic Vesicles Contain a V-Type and a P-Type ATPase

Fifty to eighty-five percent of the ATPase activity in different preparations of cholinergic synaptic vesicles isolated from Torpedo electric organ was half-inhibited by 7 microM vanadate. This activity is due to a recently purified phosphointermediate, or P-type, ATPase, Acetylcholine (ACh) active transport by the vesicles was stimulated about 35% by vanadate, demonstrating that the P-type enzyme is not the proton pump responsible for ACh active transport. Nearly all of the vesicle ATPase activity was inhibited by N-ethylmaleimide. The P-type ATPase could be protected from N-ethylmaleimide inactivation by vanadate, and subsequently reactivated by complexation of vanadate with deferoxamine. The inactivation-protection pattern suggests the presence of a vanadate-insensitive, N-ethylmaleimide-sensitive ATPase consistent with a vacuolar, or V-type, activity expected to drive ACh active transport. ACh active transport was half-inhibited by 5 microM N-ethylmaleimide, even in the presence of vanadate. The presence of a V-type ATPase was confirmed by Western blots using antisera raised against three separate subunits of chromaffin granule vacuolar ATPase I. Both ATPase activities, the P-type polypeptides, and the 38-kilodalton polypeptide of the V-type ATPase precisely copurify with the synaptic vesicles. Solubilization of synaptic vesicles in octaethyleneglycol dodecyl ether detergent results in several-fold stimulation of the P-type activity and inactivation of the V-type activity, thus explaining why the V-type activity was not detected previously during purification of the P-type ATPase. It is concluded that cholinergic vesicles contain a P-type ATPase of unknown function and a V-type ATPase which is the proton pump.     

Isolation of Escherichia coli mutants with an adenosine triphosphatase insensitive to aurovertin.

Energy-transducing adenosine triphosphatase (ATPase) from Escherichia coli is inhibited by aurovertin. Aurovertin-resistant mutants were generated by nitrosoguanidine mutagenesis of E. coli AN180, whose growth on a nonfermentable carbon source was blocked by aurovertin. The ATPase activity of cell extracts from 15 different mutants (designated MA1, MA2, MA3, etc.) was found to be at least 20 times less sensitive to aurovertin than that from the parent strain. The aurovertin-resistant mutants did not show cross-resistance towards a number of ATPase inhibitors including azide, dicyclohexylcarbodiimide, quercetin, 7-chloro-4-nitrobenzofurazan, and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. Aurovertin inhibited the energization brought about by addition of ATP to E. coli AN180 membrane vesicles; it was without effect on MA1 and MA2 membrane vesicles energized by ATP. The mutation in MA1, like other mutations of the ATPase complex, maps in the unc region of the bacterial chromosome.     

Properties and function of the proton-translocating adenosine triphosphatase of Clostridium perfringens.

Growth of Clostridium perfringens was inhibited by compounds which dissipate or prevent the formation of electrochemical proton gradients. Membrane vesicles prepared from this organism exhibited Mg2+-dependent adenosine triphosphatase (ATPase) activity sensitive to N,N'-dicyclohexylcarbodiimide. Mg2+-ATPase activity was optimal of 50 degrees C, but no discrete pH optimum was observed. Adenosine triphosphate-dependent quenching of the fluorescence of the weak base quinacrine by everted membrane vesicles suggested that the Mg2+-ATPase is a proton pump capable of generating an electrochemical proton gradient. Adenosine triphosphate-dependent transport of Ca2+ by everted vesicles was sensitive to uncouplers and inhibitors of the Mg2+-ATPase.     

The effect of F0 inhibitors on the Vibrio alginolyticus membrane ATPase.

The inhibition of membrane ATPase from the marine alkalotolerant bacterium Vibrio alginolyticus by DCCD, triphenyltin and venturicidin was studied. DCCD proved to be an irreversible inhibitor, while venturicidin and triphenyltin produced a reversible inhibitory effect. The DCCD-binding proteolipid was identified in the membrane preparations. The effect of the inhibitors on ATPase activity and ATP-dependent Na⁺-transport in V. alginolyticus subcellular vesicles is discussed.     

ATP-driven transhydrogenase provides an example of delocalized chemiosmotic coupling in reconstituted vesicles and in submitochondrial particles

The mechanism of coupling between mitochondrial ATPase (EC 3.6.1.3) and nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) was studied in reconstituted liposomes containing both purified enzymes and compared with their behavior in submitochondrial particles. In order to investigate the mode of coupling between the transhydrogenase and the ATPase by the double-inhibitor and inhibitor-uncoupler methods, suitable inhibitors of transhydrogenase and ATPase were selected. Phenylarsine oxide and A3'-O-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)-NAD+ were used as transhydrogenase inhibitors, whereas of the various ATPase inhibitors tested aurovertin was found to be the most convenient. The inhibition of the ATP-driven transhydrogenase activity was proportional to the inhibition of both the ATPase and the transhydrogenase. Inhibitor-uncoupler titrations showed an increased sensitivity of the coupled reaction towards carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)--an uncoupler that preferentially uncouples localized interactions, according to Herweijer et al. (Biochim. Biophys. Acta 849 (1986) 276-287)--when the primary pump was partially inhibited. However, when the secondary pump was partially inhibited the sensitivity towards FCCP remained unchanged. Similar results were obtained with submitochondrial particles. These results are in contrast to those obtained previously with the ATP-driven reverse electron flow. In addition, the amount of uncoupler required for uncoupling of the ATP-driven transhydrogenase was found to be similar to that required for the stimulation of the ATPase activity, both in reconstituted vesicles and in submitochondrial particles. Uncoupling of reversed electron flow to NAD+ required much less uncoupler. On the basis of these results, it is proposed that, in agreement with the chemiosmotic model, the interaction between ATPase and transhydrogenase in reconstituted vesicles as well as in submitochondrial particles occurs through the delta mu H+. In contrast, the energy transfer between ATPase and NADH-ubiquinone oxidoreductase appears to occur via a more direct interaction, according to the above-mentioned results by Herweijer et al.     

Identity and origin of the ATPase activity associated with neuronal microtubules. II. Identification of a 50,000-dalton polypeptide with ATPase activity similar to F-1 ATPase from mitochondria

We determined that the ATPase activity contained in preparations of neuronal microtubules is associated with a 50,000-dalton polypeptide by four different methods: (a) photoaffinity labeling of the pelletable ATPase fraction with [gamma-32P]-8-azido-ATP; (b) analysis of two- dimensional gels (native gel X SDS slab gel) of an ATPase fraction solubilized by treatment with dichloromethane; (c) ATPase purification by glycerol gradient sedimentation and gel filtration chromatography of a solvent-released ATPase fraction, (d) demonstration of the binding of affinity-purified antibody to the 50-kdalton polypeptide to ATPase activity in vitro. Beginning with preparations of microtubules we have purified the ATPase activity greater than 700-fold and estimate that the purified enzyme has a specific activity of 20 mumol Pi x mg-1 x min- 1 and comprises 80-90% of the total ATPase activity associated with neuronal microtubules. With affinity-purified antibody we also demonstrate cross-reactivity to the 50-kdalton subunits of mitochondrial F-1 ATPase and show that the antibody specifically labels mitochondria in PtK-2 cells. Biochemical comparisons of the enzymes reveal similar but not identical subunit composition and sensitivity to mitochondrial ATPase inhibitors. These studies indicate that the principal ATPase activity associated with microtubules is not contained in high molecular weight proteins such as dynein or MAPs and support the hypothesis that the 50-kdalton ATPase is a membrane protein and may be derived from mitochondria or membrane vesicles with F-1-like ATPase activity.     

Energy-transducing membrane-bound coupling factor-ATPase from Mycobacterium phlei. I. Purification, homogeneity, and properties.

The membrane-bound coupling factor from Mycobacterium phlei was solubilized from membrane vesicles by washing with low ionic strength buffer or 0.25 M sucrose. The solubilized enzyme exhibited coupling factor, latent ATPase, and succinate oxidation-stimulating activity. Purification by affinity chromatography using Sepharose coupled to ADP yielded a homogeneous preparation of latent ATPase which was purified about 200-fold with an 84% yield in a single step. Purified latent ATPase exhibited coupling factor activity but no succinate oxidation-stimulating activity. The molecular weight of latent ATPase was determined to be 250,000 +/- 10,000 by Sephadex G-200 chromatography. The ATPase was unmasked by trypsin treatment and activated by Mg2+ ion. However, trypsin treatment inactivated the coupling factor activity in the purified enzyme, indicating that the catalytic sites for ATPase and coupling activity are different. Unlike mitochondrial ATPase, latent ATPase from M. phlei was not cold-labile. Of the nucleoside triphosphates, UTP, ITP, and epsilon-ATP (1-N6-ethenoadenosine triphosphate) were hydrolyzed to a lesser extent compared to ATP. Kinetic data showed that ADP acted as a competitive inhibitor of latent ATPase activity with a Ki of 5 x 10(-3) M. Uncouplers of oxidative phosphorylation and respiratory inhibitors did not affect the latent ATPase activity, while sodium azide (0.1 mM) inhibited the latent ATPase activity.     

Evidence for adenylate nucleotide transport (ATP-ADP translocation) in vesicles of Frankia sp. strain EAN1pec.

Atractyloside and carboxyatractyloside partially inhibited nitrogenase activity (acetylene reduction) by isolated vesicles of Frankia strain EAN1pec. Extracts of disrupted vesicles showed nitrogenase activity that was not affected by the inhibitors. The vesicles accumulated ATP by an atractyloside-sensitive mechanism. This inhibition of ATP uptake was reversed when vesicles were permeabilized by detergent. Uptake of ATP was inhibited by excess ATP and ADP, but not AMP or adenosine, and by a calcium-dependent ATPase inhibitor. Uptake was stimulated by calcium ions. Accumulation of ATP was accompanied by release of ADP and AMP from the vesicles. The ATP taken up by vesicles and cells grown with N2 as the nitrogen source was found in the corresponding cell pools only as ATP. The data indicate activity of an ATP-ADP translocase system in vesicles of this organism. The role of ATP translocation in the symbiosis between Frankia strain EAN1pec and plant root nodules is discussed.     

Reconstitution of the purified calmodulin-dependent (Ca2+ + Mg2+)-ATPase from smooth muscle

The purified calmodulin dependent (Ca2+ + Mg2+)-ATPase (CaMg ATPase) from porcine antral smooth muscle transports Ca2+ after reconstitution in lipid vesicles indicating that this enzyme is indeed a Ca2+-transport ATPase. For CaMg ATPase reconstituted in asolectin vesicles a good correlation was found between the time course of Ca2+ accumulation and the corresponding changes in CaMg ATPase activity. The ATPase activity was stimulated 8-fold by A23187, which further indicates a tight coupling between ATP hydrolysis and Ca2+ transport. Asolectin vesicles with incorporated enzyme accumulated Ca2+ with a ratio approaching one Ca2+ ion transported for each ATP hydrolyzed. For CaMg ATPase reconstituted in phosphatidylcholine vesicles on the other hand, Ca2+ transport and CaMg ATPase were poorly coupled as is shown by the approximately 3.5 fold stimulation by A23187. The activity of the CaMg ATPase when reconstituted in asolectin vesicles was stimulated 1.25 fold by calmodulin while in phosphatidylcholine a value of 4.25 was obtained. The CaMg ATPase activity of the enzyme reconstituted either in asolectin or phosphatidylcholine was, after its stimulation by A23187, still further stimulated by detergent by a factor of 5.     

Orientation of membrane vesicles fromEscherichia coli prepared by different procedures

Summary The orientation of membrane vesicles prepared fromEscherichia coli by either French press, sonication or ethylenediamine tetraacetate (EDTA)-lysozyme was examined. The following procedures were used to determine orientation: (1) accessibility of the impermeable ferricyanide ion to the respiratory chain; (2) inhibition of membranal ATPase by specific antiserum; (3) binding of ATPase to the membrane. Data with spheroplasts indicated that ATPase, ATPase binding sites and ferricyanide reductase activities were localized on the inner part of the cytoplasmic membrane. Thus, there was no demonstrable NADH-ferricyanide reductase activity, low ATPase activity, no inhibition of ATPase by antiserum and no binding of purified ATPase by spheroplasts. In the case of membrane vesicles prepared by French press or sonication, the ATPase activity, the ATPase binding site and the site where ferricyanide takes electrons from the respiratory chain all appeared to be on the outside of the vesicles, suggesting that they are inverted. In the case of EDTA-lysozyme vesicles, which are widely used for transport studies, about half of the ATPase binding sites and ferricyanide reactive sites were exposed to the outside. Sixty percent of the ATPase activity was sensitive to antiserum. The two most probable explanations for these data are: (1) partial inversion of EDTA-lysozyme vesicles in the course of preparation; (2) movement of marker enzymes within the membrane vesicles during their isolation.     

Uncoupling of Cholinergic Synaptic Vesicles by the Presynaptic Toxin β-Bungarotoxin

The effect of the presynaptic neurotoxin beta-bungarotoxin (beta-BuTx) on the acetylcholine (ACh) storage system of synaptic vesicles isolated from the electric organ of Torpedo californica was studied. The toxin can totally inhibit active transport of [3H]ACh by the vesicles in a Ca2+-, time-, and concentration-dependent manner. Correlated with these effects is a 50-60% stimulation of the vesicle proton-pumping ATPase activity. The beta-BuTx-mediated transport inhibition and ATPase stimulation are antagonized by delipidated bovine serum albumin, not reversed by excess EGTA, and not mimicked by other cationic proteins or soybean or pancreatic trypsin inhibitors. The behavior is consistent with phospholipase A2 (PLA2)-dependent damage to the vesicle membrane caused by beta-BuTx, which results in uncoupling of the ATPase and ACh transporter systems. The nonneurotoxic Naja naja venom PLA2 causes similar effects, except that it is slightly more potent on a molar basis. About 100-fold more beta-BuTx is required to effect lysis of synaptic vesicles than to uncouple them. ATP is a strong inhibitor of beta-BuTx- but not of N. naja PLA2-mediated uncoupling. The observations suggest that a component of beta-BuTx toxicity in the cholinergic terminal might involve attack on synaptic vesicles or vesicle-like structures and that a nucleotide-like factor might modulate the toxicity.     

Charged Amino Acids in a Preprotein Inhibit SecA-Dependent Protein Translocation

Sec translocase catalyzes membrane protein insertion and translocation. We have introduced stretches of charged amino acid residues into the preprotein proOmpA and have analyzed their effect on in vitro protein translocation into Escherichia coli inner membrane vesicles. Both negatively and positively charged amino acid residues inhibit translocation of proOmpA, yielding a partially translocated polypeptide chain that blocks the translocation site and no longer activates preprotein-stimulated SecA ATPase activity. Stretches of positively charged residues are much stronger translocation inhibitors and suppressors of the preprotein-stimulated SecA ATPase activity than negatively charged residues. These results indicate that both clusters of positively and negatively charged amino acids are poor substrates for the Sec translocase and that this is reflected by their inability to stimulate the ATPase activity of SecA.     

The effect of F0 inhibitors on the Vibrio alginolyticus membrane ATPase

The inhibition of membrane ATPase from the marine alkalotolerant bacterium Vibrio alginolyticus by DCCD, triphenyltin and venturicidin was studied. DCCD proved to be an irreversible inhibitor, while venturicidin and triphenyltin produced a reversible inhibitory effect. The DCCD-binding proteolipid was identified in the membrane preparations. The effect of the inhibitors on ATPase activity and ATP-dependent Na⁺-transport in V. alginolyticus subcellular vesicles is discussed.     

The role of the carbodiimide-reactive component of the adenosine-5'-triphosphatase complex in the proton permeability of Escherichia coli membrane vesicles.

Membrane vesicles isolated from wild-type and dicyclohexylcarbodiimide-resistant strains of Escherichia coli exhibit identical respiration-dependent transport activities, and in both cases, this activity is abolished by extraction of the vesicles with 1.0 M guanidine-HCl. Transport activity of extracted wild-type vesicles is completely restored by exposing the vesicles to lipophilic or water-soluble carbodiimides, while transport activity of the mutant vesicles is not restored by exposure to lipophilic carbodiimides. Strikingly, however, complete reactivation of transport in mutant vesicles is observed with water-soluble carbodiimides. Similarly, the Ca2+, Mg2+-stimulated ATPase activity of wild-type vesicles is inhibited by both classes of carbodiimides, while the ATPase activity of mutant vesicles is inhibited by water-soluble carbodiimides, but resistant to inhibition by lipophilic carbodiimides. The carbodiimide-reactive component of the membraneous Ca2+, Mg2+-stimulated ATPase complex in wildtype vesicles is readily labeled with N,N'-dicyclohexyl[14C]-carbodiimide, while the analogous component in mutant vesicles is not reactive. Alternatively, when vesicles are treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [14C]methiodide, a water-soluble carbodiimide, the carbodiimide-reactive component is labeled to a similar degree in both preparations. The results suggest that the altered carbodiimide-reactive proteolipid in the dicyclohexylcarbodiimide-resistant mutant is specifically defective in its ability to react with lipophilic carbodiimides. In addition, these and other findings indicate that the increase in proton permeability observed on extraction of isolated membrane vesicles with chaotropic agents is due exclusively to an effect on the carbodiimide-reactive component of the Ca2+, Mg2+-stimulated ATPase complex.     

Sidedness of (Calcium, Magnesium) Adenosine Triphosphatase of Purified Torpedo Synaptic Vesicles

Abstract: Purified Torpedo synaptic vesicles contain ouabain-insensitive Mg²⁺_τ and Ca²⁺-stimulated ATPase activity. The sidedness of the ATPase on the vesicular membranes was investigated. Addition of ATP and Mg²⁺ or Ca²⁺ to intact vesicles results in activation of the ATPase. Exposure of the vesicles to low concentrations of Lubrol-PX and Triton X-100, which do not solubilize the activity, results in the concurrent release of the vesicular contents and in an increase of the Mg²⁺-ATPase activity, whereas the Ca²⁺-dependent activity is drastically decreased. p -Chloromercuribenzene sulphonate (PCMBS) almost completely inhibits the activity of detergent-treated vesicles whereas that of the native material is only slightly affected. Tryptic digestion of intact vesicles and of vesicular ghosts results in partial reduction of the ATPase activity. These results suggest that the vesicles contain an outward oriented Ca²⁺/Mg²⁺ ATPase activity which can be modulated by detergents.