Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The potential toxicity of the herbicide paraquat (1,1-dimethyl-4,4'-bipyridylium dichloride) was tested in bioenergetic functions of isolated rat liver mitochondria. Paraquat increases the rate of State 4 respiration, doubling at 10 mM, indicating uncoupling effects. Additionally, State 3 respiration is depressed by about 15%, at 10 mM paraquat, whereas uncoupled respiration in the presence of CCCP is depressed by about 30%. Furthermore, paraquat partially inhibits the ATPase activity through a direct effect on this enzyme complex. However, at high concentrations (5-10 mM), the ATPase activity is stimulated, probably as consequence of the described uncoupling effect. Depression of respiratory activity is mediated through partial inhibitions of mitochondrial complexes III and IV. Paraquat depresses delta psi as a function of herbicide concentration. In addition, the depolarization induced by ADP is decreased and repolarization is biphasic suggesting a double effect. Repolarization resumes at a level consistently higher than the initial level before ADP addition, for paraquat concentrations up to 10 mM. This particular effect is clear at 1 mM paraquat and tends to fade out with increasing concentrations of the herbicide.
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PMID:Mitochondrial bioenergetics is affected by the herbicide paraquat. 772 98

Ca2+ transport has been measured across plasma membrane vesicles isolated from cells of Ustilago maydis. This transport was found to be ATP- (or to a lesser extent GTP) and Mg(2+)-dependent. Inconsistent release of Ca2+ from intact vesicles was obtained using the calcium ionophore A23187. However, Ca2+ was released by Triton X-100 in a concentration-dependent manner. Transport was inhibited by vanadate (> 50%) and erythrosin B (about 50%), I50 being about 10 microM for both inhibitors. In the presence of the protonophores CCCP or gramicidin, partial inhibition of Ca2+ transport (about 20%) was observed, but the Ca(2+)-channel blockers, nifedipine, diltiazem and verapamil had no effect, although the latter inhibited proton transport. The results indicate that Ca2+ transport in U. maydis is regulated by a P-type ATPase with similar properties to that found in higher plants.
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PMID:Ca(2+)-ATPase-driven calcium accumulation in Ustilago maydis plasma membrane vesicles. 781 45

Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence has implicated either the trans-Golgi network (TGN) or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin, proSRIF, (Stoller TJ, Shields D (1988) J Cell Biol 107, 2087-2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident. Following the 20 degrees C block, the cells were permeabilized and proSRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by pre-incubating the permeabilized cells with low concentrations of N-ethylmaleimide or bafilomycin, both inhibitors of vacuolar-type ATP-dependent proton pumps. These data suggest that ATP is required for generation of an acidic pH in the lumen of the TGN which is necessary for the activity of prohormone processing enzymes. By exploiting a permeabilized cell system, we have demonstrated that proSRIF cleavage is initiated in the TGN, in a reaction which is facilitated by a Golgi-associated vacuolar type ATPase.
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PMID:Prohormone processing in permeabilized cells: endoproteolytic cleavage of prosomatostatin in the trans-Golgi network. 781 32

The aim of this work was to examine the effects of changes in external K+ concentration (Ko) around its physiological value, of various K+ channels blockers, including internal Cs+, of vacuolar H(+)-ATPase inhibitors and of the protonophore CCCP on the resting potential and the voltage-dependent K+ current of differentiated neuroblastoma x glioma hybrid NG108-15 cells using the whole-cell patch-clamp technique. The results are as follows: (i) under standard conditions (Ko = 5 mM) the membrane potential was -60 +/- 1 mV. It was unchanged when Ko was decreased to 1 mM and was depolarized by 4 +/- 1 mV when Ko was increased to 10 mM. (ii) Internal Cs+ depolarized the membrane by 21 +/- 3 mV. (iii) The internal application of the vacuolar H(+)-ATPase inhibitors N-ethylmaleimide (NEM), NO3- and bafilomycin A1 (BFA) depolarized the membrane by 15 +/- 2, 18 +/- 2 and 16 +/- 2 mV, respectively. (iv) When NEM or BFA were added to the internal medium containing Cs+, the membrane was depolarized by 45 +/- 1 and 42 +/- 2 mV, respectively. (v) The external application of CCCP induced a transient depolarization followed by a prolonged hyperpolarization. This hyperpolarization was absent in BFA-treated cells. The voltage-dependent K+ current was increased at negative voltages and decreased at positive voltages by NEM, BFA and CCCP. Taken together, these results suggest that under physiological conditions, the resting potential of NG108-15 neuroblastoma cells is maintained at negative values by both voltage-dependent K+ channels and an electrogenic vacuolar type H(+)-ATPase.
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PMID:Contribution of a H+ pump in determining the resting potential of neuroblastoma cells. 800 50

Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin (proSRIF) (Stoller, T. J., and D. Shields. J. Cell Biol. 1988. 107:2087-2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37 degrees C. After the 20 degrees C block, the cells were mechanically permeabilized and pro-SRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by preincubating the permeabilized cells with low concentrations of N-ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps. These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occurred at 20 degrees C; in contrast, at 37 degrees C, the budding of secretory vesicles was approximately 40% efficient and was dependent on ATP, GTP, and cytosolic factors. Vesicle formation was inhibited by GTP gamma S suggesting a role for GTP-binding proteins in this process. Vesicle budding was dependent on cytosolic factors that were tightly membrane associated and could be removed only by treating the permeabilized cells with high salt. After high salt treatment, vesicle formation was dependent on added cytosol or the dialyzed salt extract. The formation of nascent secretory vesicles contrasts with prosomatostatin processing which required only ATP for efficient cleavage. Our results demonstrate that prohormone cleavage which is initiated in the TGN, precedes vesicle formation and that processing can be uncoupled from the generation of nascent secretory vesicles.
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PMID:Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells. 810 89

Cisplatin (CP) nephrotoxicity in vivo is characterized by proximal tubule (PT) and collecting duct dysfunction. We reported previously that mitochondrial injury is an important early event in CP toxicity to PT cells and precedes inhibition of Na+,K(+)-ATPase activity and loss of cell K+. In the present study, we monitored oxygen consumption (QO2) and net K+ fluxes in intact inner medullary collecting duct (IMCD) and PT cells in vitro, using O2- and K(+)-sensitive electrodes, to determine if CP has similar effects on IMCD cells. Short-term exposure of IMCD cells to CP resulted in inhibition of spontaneous, ouabain-sensitive and ouabain-oversensitive QO2, but to a lesser degree than in PT. Ouabain-sensitive K+ transport and cell K+ content were also reduced in intact IMCD cells in this setting, confirming inhibition of Na+,K(+)-ATPase activity. In contrast, Na+,K(+)-ATPase activity measured in IMCD cell lysates was not altered. These results suggested that CP inhibited Na+,K(+)-ATPase activity in intact IMCD cells indirectly either by blocking Na+ entry or by inhibiting mitochondrial oxidative phosphorylation. Nystatin (Na+ ionophore) and carbonyl cyanide m-chlorophenylhydrazone (CCCP, uncoupler of oxidative phosphorylation) were used to distinguish between these possibilities. Nystatin-stimulated and CCCP-uncoupled QO2 were reduced in CP-treated IMCD cells by 34 +/- 10% and 25 +/- 5%, respectively, indicating mitochondrial injury. Again, the effects of CP on nystatin-stimulated and CCCP-uncoupled QO2 in IMCD cells were significantly less dramatic than in PT cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential actions of cisplatin on renal proximal tubule and inner medullary collecting duct cells. 838 66

In inverted subcellular vesicles of Escherichia coli grown at high delta mu H+ (neutral pH, no protonophorous uncoupler), ATP-driven Na+ transport and oxidative phosphorylation are completely inhibited by the protonophore CCCP. If E. coli was grown at low delta mu H+, i.e. at high pH or in the presence of uncoupler, some oxidative phosphorylation was observed in the vesicles even in CCCP-containing medium, and Na+ transport was actually stimulated by CCCP. The CCCP-resistant transport and phosphorylation were absent from the unc mutant lacking F0F1 ATPase. Both processes proved to be sensitive to (i) the Na+/H+ antiporter monensin, (ii) the Na+ uniporter ETH 157, (iii) the F0 inhibitors DCCD and venturicidin, and (iv) the F1 inhibitor aurovertin. The CCCP-resistant oxidative phosphorylation was stimulated by Na+ and arrested by oppositely directed delta pNa. These data are consistent with the assumption that, under appropriate growth conditions, the F0F1-type ATPase of E. coli becomes competent in transporting Na+ ions.
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PMID:ATP-driven Na+ transport and Na(+)-dependent ATP synthesis in Escherichia coli grown at low delta mu H+. 842 16

The characteristics of L-proline transport in the procyclic form of Trypanosoma brucei were studied by using L-[14C]proline and a quick separation technique by centrifugation through an oil mixture. L-Proline uptake displayed typical Michaelis-Menten kinetics, with a Km of 19 microM and a maximum transport velocity of 17 nmol/min per 10(8) cells at 27 degrees C. The maximum concentration gradient factor obtained after 1 min of incubation was 270-fold in 0.02 mM proline. Cells permeabilized with 80 microM digitonin were still able to accumulate 14C label, but to a lower extent. The temperature-dependence of proline uptake gave an apparent activation energy of 74.9 kJ.mol-1. In competition studies with a 10-fold excess of structural analogues, L-alanine, L-cysteine and L-azetidine-2-carboxylate were found to inhibit L-proline uptake. Variation of pH or addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone ('CCCP') did not affect proline transport, showing that it is not driven by a protonmotive force. The absence of Na+, with or without monensin, did not affect proline transport. The absence of K+ and the addition of the Na+,K(+)-ATPase inhibitor ouabain had no significant effect on proline uptake activity. The thiol-modifying reagent iodoacetate (10 mM) decreased proline uptake by half. KCN (1 mM) inhibited proline uptake to a lesser extent, and the degree of inhibition was proportional to the intracellular ATP concentration. Preliminary experiments on proline transport in plasma-membrane vesicles of the cells, using a filtration technique, showed an uptake of proline (0.67 nmol/mg of protein) by the vesicles, but only in the presence of intravesicular ATP. The results thus obtained suggest that the proline carrier system in T. brucei is ATP-driven and independent of Na+, K+ or H+ co-transport.
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PMID:Active transport of L-proline in the protozoan parasite Trypanosoma brucei brucei. 847 Oct 48

Contrasting observations exist which indicate that in plants the fluorescent dye lucifer yellow CH (LYCH) either can be used as a tracer for endocytosis or as a substrate for an anion transporter located at the vacuolar membrane. In addition, LYCH as a disulphonated substance may represent an analogue of sulphonated or sulfated natural compounds like some flavonoids. We performed uptake experiments with LYCH into isolated rye vacuoles and observed saturable (Km = 0.3-0.6 mM) vacuolar transport and accumulation of the dye against the concentration gradient only when MgATP was present. GTP and, to a low extent, UTP could substitute for ATP, while the non-hydrolysable ATP analogue AMP-PNP did not drive LYCH uptake. Vanadate and probenecid, the latter substance is known to inhibit organic anion transport at the liver canalicular membrane, both strongly decreased the vacuolar uptake of LYCH, while bafilomycin A1, a specific inhibitor of the vacuolar H+-ATPase, had no effect. Together with the fact that abolishment of the delta pH via CCCP had only a weak influence on LYCH accumulation, our results indicate that this compound is taken up into rye vacuoles by a directly energized process. Uptake of LYCH was strongly inhibited by other sulfated compounds including sulfobromophthalein and the flavones apigenin 7,4'-disulfate and luteolin 7,4'-disulfate arguing for the presence of a vacuolar transporter for structurally different sulphonated or sulfated compounds. Glucuronates like the rye-specific flavone luteolin 7-O-diglucuronide also strongly decreased uptake of the dye, whereas only a weak effect was observed in the presence of glutathione and a glutathione conjugate, suggesting that LYCH uptake is not mediated via the vacuolar glutathione conjugate pump.
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PMID:Transport of lucifer yellow CH into plant vacuoles--evidence for direct energization of a sulphonated substance and implications for the design of new molecular probes. 945 May 55

The kinetics of adenosine triphosphate (ATP)-dependent proton transport into clathrin-coated vesicles from bovine brain have been studied. We observe that the vacuolar proton-translocating ATPase (V-ATPase) from clathrin-coated vesicles is subject to two different types of inhibition by ADP. The first is competitive inhibition with respect to ATP, with a Ki for ADP of 11 microM. The second type of inhibition occurs after preincubation of the V-ATPase in the presence of ADP and Mg2+, which results in inhibition of the initial rate of proton transport followed by reactivation over the course of several minutes. The second effect is observed at ADP concentrations as low as 0.1-0.2 microM, indicating that a high affinity inhibitory complex is formed between ADP and the V-ATPase and is only slowly dissociated after the addition of ATP. We have further investigated the effect of sodium azide, an inhibitor of the F-ATPases that has been shown to stabilize an inactive complex between ADP and the F1-F0-ATP synthase (F-ATPase). We observed that azide inhibited ATP-dependent proton transport by the purified, reconstituted V-ATPase with a K0.5 of 0.2-0.4 mM but had no effect on ATP hydrolysis. Azide was shown not to increase the passive proton permeability of reconstituted vesicles and did not stimulate ATP hydrolysis by the reconstituted enzyme, in contrast with CCCP, which both abolished the proton gradient and stimulated hydrolysis. Thus, azide does not appear to act as a simple uncoupler of proton transport and ATP hydrolysis. Rather, azide may have some more direct effect on V-ATPase activity. Possible mechanisms by which azide could exert this effect on the V-ATPase and the contrasting effects of azide on the F- and V-ATPases are discussed.
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PMID:Interaction of the clathrin-coated vesicle V-ATPase with ADP and sodium azide. 972 93


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