Inhibition of Escherichia coli H+-ATPase by venturicidin, oligomycin and ossamycin

The antibiotics venturicidin, oligomycin and ossamycin were investigated as potential inhibitors of the Escherichia coli H+-ATPase. It was found that venturicidin strongly inhibited ATP-driven proton transport and ATP hydrolysis, while oligomycin weakly inhibited these functions. Inhibition of the H+-ATPase by venturicidin and oligomycin was correlated with inhibition of F0-mediate proton transport. Both inhibitors were found to interfere with the covalent reaction between dicyclohexyl[14C]carbodiimide and the F0 subunit c (uncE protein). Ossamycin had no direct inhibitory effect on E. coli F0 or F1; rather, it was found to uncouple ATP hydrolysis from proton transport.     

Inhibition of gastric (H+ + K+)-ATPase by unsaturated long-chain fatty acids

Arachidonic acid and unsaturated C18 fatty acids at concentrations near 10(-5) M markedly inhibited (H+ + K+)-ATPase in hog or rat gastric membranes. Arachidonic acid was a more potent inhibitor than unsaturated C18 fatty acids, but the involvement of the metabolites of arachidonic acid cascade was ruled out. Linolenic acid inhibited the formation of phosphoenzyme and the K+ -dependent p-nitrophenylphosphatase activity of the hog ATPase. Treatment with fatty acid-free bovine serum albumin abolished only the inhibitory effect of the fatty acid on the phosphatase activity without restoring the overall ATPase action. These data suggest the existence of at least two groups of hydrophobic binding sites in the gastric ATPase for unsaturated long-chain fatty acids which affect differentially the catalytic reactions of the ATPase. (H+ + K+)-ATPase in rat gastric membranes was found more susceptible to the fatty acid inhibition and also more unstable than the ATPase in hog gastric membranes. The presence of a millimolar level of lanthanum chloride or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid stabilized the rat ATPase probably via the inhibition of Ca2+ -dependent phospholipases in the gastric membranes.     

Inhibition of Host Vacuolar H+-ATPase Activity by a Legionella pneumophila Effector

Legionella pneumophila is an intracellular pathogen responsible for Legionnaires'' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae.     

Inhibition of gastric (H+ + K+)-ATPase by the substituted benzimidazole, picoprazole

The substituted benzimidazole, picoprazole, inhibited the gastric (H+ + K+)-ATPase in a concentration-and time-dependent manner. Half-maximal inhibition of the (H+ + K+)-ATPase activity was obtained at about 2 . 10(-6)M under standard conditions. In addition to the inhibition of ATPase activity, parallel inhibition of phosphoenzyme formation and the proton transport activity were achieved. Radiolabelled picoprazole was found to bind to 100 kDa peptide; this peptide was shown by phosphorylation experiments to contain the catalytic centre of the (H+ + K+)-ATPase. Studies on the (Na+ + K+)-ATPase indicated that this enzyme was unaffected by picoprazole. From the data presented and from other pharmacological studies, it is proposed that this compound inhibits acid secretion at the level of the parietal cell by its ability to inhibit the gastric proton pump, the (H+ + K+)-ATPase.     

Mode of Cell Death Induction by Pharmacological Vacuolar H+-ATPase (V-ATPase) Inhibition

The vacuolar H⁺-ATPase (V-ATPase), a multisubunit proton pump, has come into focus as an attractive target in cancer invasion. However, little is known about the role of V-ATPase in cell death, and especially the underlying mechanisms remain mostly unknown. We used the myxobacterial macrolide archazolid B, a potent inhibitor of the V-ATPase, as an experimental drug as well as a chemical tool to decipher V-ATPase-related cell death signaling. We found that archazolid induced apoptosis in highly invasive tumor cells at nanomolar concentrations which was executed by the mitochondrial pathway. Prior to apoptosis induction archazolid led to the activation of a cellular stress response including activation of the hypoxia-inducible factor-1α (HIF1α) and autophagy. Autophagy, which was demonstrated by degradation of p62 or fusion of autophagosomes with lysosomes, was induced at low concentrations of archazolid that not yet increase pH in lysosomes. HIF1α was induced due to energy stress shown by a decline of the ATP level and followed by a shutdown of energy-consuming processes. As silencing HIF1α increases apoptosis, the cellular stress response was suggested to be a survival mechanism. We conclude that archazolid leads to energy stress which activates adaptive mechanisms like autophagy mediated by HIF1α and finally leads to apoptosis. We propose V-ATPase as a promising drugable target in cancer therapy caught up at the interplay of apoptosis, autophagy, and cellular/metabolic stress.     

H+ transport by reconstituted gastric (H+ + K+)-ATPase

Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol liposomes by means of a freeze-thaw-sonication technique. Upon addition of MgATP, active H+ transport was observed, with a maximal rate of 2.1 mumol X mg-1 X min-1, requiring the presence of 100 mM K+ at the intravesicular site. However, in the absence of ATP an H+-K+ exchange with a maximal rate of 0.12 mumol X mg-1 X min-1 was measured, which could be inhibited by the well-known ATPase inhibitors vanadate and omeprazole, giving the first evidence of a passive K+-H+ exchange function of gastric (H+ + K+)-ATPase. An Na+-H+ exchange activity was also measured, which was fully inhibited by 1 mM amiloride. Simultaneous reconstitution of Na+/H+ antiport and (H+ + K+)-ATPase could explain why reconstituted ATPase appeared less cation-specific than the native enzyme (Rabon, E.C., Gunther, R.B., Soumarmon, A., Bassilian, B., Lewin, M.J.M. and Sachs, G. (1985) J. Biol. Chem. 260, 10200-10212).     

Inhibition of (H+ + K+)-ATPase by omeprazole in isolated gastric vesicles requires proton transport

Omeprazole was found to inhibit the (H+ + K+)-ATPase activity in isolated gastric vesicles only when acid was accumulated in the vesicle lumen. The ATPase activity was time- and dose-dependently inhibited in the presence of K+ and valinomycin. Under conditions in which no pH-gradient was generated, i.e., in the presence of K+ alone or NH4+, no effect of omeprazole was found. The degree of inhibition was directly correlated to the amount of inhibitor bound to the preparation. A stoichiometry of 2 mol radiolabelled inhibitor bound per mol phosphoenzyme was found on total inhibition of the K+ plus valinomycin-stimulated activity. This inhibitory action of omeprazole on the ATPase activity could be fully reversed by addition of beta-mercaptoethanol. The inhibition of the proton transport in the (H+ + K+)-ATPase-containing vesicles by omeprazole was also strictly correlated to the amount of bound inhibitor. The stoichiometry of binding at total inhibition of this reaction was found to be 1.4 mol per mol phosphoenzyme. The K+-stimulated p-nitrophenylphosphatase activity was inhibited in parallel with the ATPase activity, whereas the phosphoenzyme levels were affected to a lesser extent by omeprazole. Gel electrophoresis of an omeprazole-inhibited vesicle preparation showed that the radiolabel was mainly found at 94 kDa, the molecular weight of the (H+ + K+)-ATPase catalytic subunit(s).     

Medium Acidification by Maize Root Tips and its Inhibition by Heavy Water

Acidification of external solution by maize root tips can apparently proceed by two mechanisms. The first, significant only above pH 6, does not require the presence of salts in the medium and appears to be due to respiratory CO₂ release with subsequent hydration and dissociation into H⁺ + HCO^?₃. The second, much more efficient process, requires permeant ions and proceeds to well below pH 4 under mediation by the plasma membrane H⁺-ATPase. While the first process is affected by acetazolamide (an inhibitor of carbonate dehydratase), the second is blocked by vanadate, erythrosin B, diethylstilbestrol and miconazole, and is strikingly stimulated by fusicoccin. Both mechanisms are markedly inhibited by heavy water, the first by up to 80% with an ID₅₀ of 18–25% D₂O, the second up to 91%, with an ID₅₀ of 20–30% D₂O. The inhibition of the acidification by ATPase can be relieved by replacing D₂O with H₂O, indicating that the effect of D₂O is kinetic rather than on covalently-bound hydrogen atoms. Apparently the H⁺-ATPase uses H⁺ (or possibly H₃O⁺) as substrate which binds to a rather specific site where it cannot be functionally replaced by D⁺ or D₃O⁺.     

Inhibition of calmodulin-activated Ca2+-ATPase by propranolol and nadolol

Propranolol, at concentrations ranging from 0.05 to 0.5 mM, inhibits the calmodulin-activated Ca2+-ATPase of human erythrocyte membranes. In the same concentration range it is without effect on the basal Ca2+-ATPase. The inhibition is competitive and appears to be due to membrane binding, rather than to combination with cytoplasmic calmodulin as is the case for phenothiazines. This effect of propranolol may explain its ability to open the calcium-gated potassium channel, and could also be related to its action as a beta-adrenergic blocker. Nadolol, another beta-adrenergic blocker, is also an inhibitor of calmodulin-activated Ca2+-ATPase.     

Inhibition of red cell Ca2+-ATPase by vanadate

1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate. 2. Several natural activators of Ca2+-ATPase (Mg2+, K+, Na+ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate. 3. Among the ligands tests, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K 1/2 for vanadate was 1.5 microM and inhibition was nearly complete at saturating vanadate concentrations. 5. There are similarities between the kinetics of inhibition of red cell Ca2+-ATPase and (Na+ + K+)-ATPase prepared from a variety of sources; however, (Na+ + K+)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by miconazole

The inhibition of sarcoplasmic reticulumCa²⁺-ATPase activity by miconazole was dependent on theconcentration of ATP and membrane protein. Half-maximal inhibition wasobserved at 12 µM miconazole when the ATP concentration was 50 µMand the membrane protein was 0.05 mg/ml. When ATP was 1 mM, a lowmicromolar concentration of miconazole activated the enzyme, whereashigher concentrations inhibited it. A qualitatively similar responsewas observed when Ca²⁺ transport was measured. Likewise,the half-maximal inhibition value was higher when the membraneconcentration was raised. Phosphorylation studies carried out aftersample preequilibration in different experimental settings shed lighton key partial reactions such as Ca²⁺ binding and ATPphosphorylation. The miconazole effect on Ca²⁺-ATPaseactivity can be attributed to stabilization of theCa²⁺-free enzyme conformation giving rise to a decrease inthe rate of the Ca²⁺ binding transition. The phosphoryltransfer reaction was not affected by miconazole.

     

Inhibition of cell growth by bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase

Summary Bafilomycin A₁, a potent selective inhibitor of vacuolar H⁺-ATPase, inhibited the growth of a variety of cultured cells dose-dependently, including golden hamster embryo and NIH-3T3 fibroblasts, whether or not they were transformed, and PC12 and HeLa cells. The concentration of bafilomycin A₁ for 50% inhibition of cell growth ranged from 10 to 50 nM. The dose response was nearly parallel with that of the bafilomycin A₁-induced lysosomal pH increase. The degree of pH increase for growth inhibition produced by bafilomycin A₁ was similar to that produced by NH₄Cl in which little difference was recognized in effect among cell types.     

Cercospora beticola Toxins (X. Inhibition of Plasma Membrane H+-ATPase by Beticolin-1)

Influxes of 13NH4+ across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Two kinetically distinct uptake systems for NH4+ were identified. In N-deprived plants, a Michaelis-Menten-type high-affinity transport system (HATS) operated in a 2.5 to 350 [mu]M range of external NH4+ concentration ([NH4 +]o). The Vmax of this HATS was 1.9 to 2.4 [mu]mol g-1 h-1, and the Km was 20 to40 [mu]M. At [NH4+]o from 500 [mu]M to 50 mM, a linear low-affinity system (LATS) was apparent. Both HATS and LATS were constitutive. A time-dependence study of NH4+ influx in previously N-deprived seedlings revealed a small transient increase of NH4+ influx after 24 h of exposure to 100 [mu]M [NH4+]o. This was followed by a decline of influx to a steady-state value after 4 d. In seedlings exposed to 100 [mu]M external NO3- concentration for 3 d, the Vmax for NH4+ uptake by HATS was increased approximately 30% compared to that found in N-deprived seedlings, whereas LATS was down-regulated. The present study defines the much higher uptake capacity for NH4+ than for N03- in seedlings of this species.     

Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction

Vacuolar H⁺-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC₅₀ of ∼1.2 μm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis.     

Production of Nitric Oxide and Phosphatidic Acid is Involved in Activation of Plasma Membrane H+-ATPase in Maize Root Tips in Simulated Drought Stress

Russian Journal of Plant Physiology - Nitric oxide (NO), phospholipase D (PLD) and plasma membrane (PM) H+-ATPase play crucial roles in plant response to drought stress, but the mechanisms of their...     

Structure and function of H+-ATPase

(1) Extensive studies on proton-translocating ATPase (H⁺-ATPase) revealed that H⁺-ATPase is an energy transforming device universally distributed in membranes of almost all kinds of cells. (2) Crystallization of the catalytic portion (F₁) of H⁺-ATPase showed that F₁ is a hexagonal molecule with a central hole. The diameter of F₁ is about 90 Å and its molecular weight is about 380,000. (3) Use of thermophilic F₁ permits the complete reconstitution of F₁ from its five subunits (, , , , and ) and demonstration of the gate function of the -complex, the catalytic function of (supported by and ), and the H⁺-translocating functions of all five subunits. (4) Studies using purified thermostable F₀ showed that F₀ is an H⁺-channel portion of H⁺-ATPase. The direct measurement of H⁺-flux through F₀, sequencing of DCCD-binding protein, and isolation of F₁-binding protein are described. (5) The subunit stoichiometry of F₁ may be ₃₃. (6) Reconstitution of stable H⁺-ATPase-liposomes revealed that ATP is directly synthesized by the flow of H⁺ driven by an electrochemical potential gradient and that H⁺ is translocated by ATP hydrolysis. This rules out functions for all the hypothetical components that do not belong to H⁺-ATPase in H⁺-driven ATP synthesis. The roles of conformation change and other phenomena in ATP synthesis are also discussed.     

Plasma Membrane H+-ATPase in Maize Roots Induced for NO3- Uptake

Plasma membrane H+-ATPase was studied in maize (Zea mays L.) roots induced for NO3- uptake. Membrane vesicles were isolated by means of Suc density gradient from roots exposed for 24 h either to 1.5 mM NO3- or 1.5 mM SO4-. The two populations of vesicles had similar composition as shown by diagnostic inhibitors of membrane-associated ATPases. However, both ATP-dependent intravesicular H+ accumulation and ATP hydrolysis were considerably enhanced (60-100%) in vesicles isolated from NO3--induced roots. Km for Mg:ATP and pH dependency were not influenced by NO3- treatment of the roots. ATP hydrolysis in plasma membrane vesicles for both control and NO3--induced roots was not affected by 10 to 150 mM NO3- or Cl-. On the other hand, kinetics of NO3-- or Cl--stimulated ATP-dependent intravesicular H+ accumulation were modified in plasma membrane vesicles isolated from NO3-- induced roots. Immunoassays carried out with polyclonal antibodies against plasma membrane H+-ATPase revealed an increased steady-state level of the enzyme in plasma membrane vesicles isolated from NO3--induced roots. Results are consistent with the idea of an involvement of plasma membrane H+-ATPase in the overall response of roots to NO3-.     

Diamide blocks H+ conductance in mitochondrial H+-ATPase by oxidizing FB dithiol

Effects of diamide on proton conductance of electron transport particles (ETPH), purified H+-ATPase (F1-F0), F0 of the H+-ATPase from beef heart mitochondria and binding of cadmium (109Cd) to the H+-ATPase have been examined in the present paper. When ETPH and purified H+-ATPase are treated with 1 mM diamide, ATP-dependent generation of membrane potential, monitored by the absorbance change produced by the redistribution of oxonol VI, is consistently inhibited. Diamide also blocks passive H+ conductance driven by a K+ diffusion potential in the membrane sector, F0, of H+-ATPase. Furthermore, diamide treatment drastically reduces the binding of 109Cd2+ to H+-ATPase, showing competition for the FB dithiol group.