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)

Addition of glucose and other sugars to derepressed cells of the fungus Fusarium oxysporum var. lini triggered activation of the plasma membrane H(+)-ATPase within 5 min. Glucose was the best activator while galactose and lactose had a lesser effect. The activation was not prevented by previous addition of cycloheximide and it was fully reversible when the glucose was removed. The activation process in vivo also caused changes in the kinetic properties of the enzyme. The non-activated enzyme had an apparent Km of about 3.2 mM for ATP whereas the activated enzyme showed an apparent Km of 0.26 mM. In addition, the pH optimum of the H(+)-ATPase changed from 6.0 to 7.5 upon activation. The activated enzyme was more sensitive to inhibition by vanadate. When F. oxysporum was cultivated in media containing glucose as the major carbon source, enhanced H(+)-ATPase activity was largely confined to the period corresponding to the lag phase, i.e. just before the start of acidification of the medium. This suggests that the activation process might play a role in the onset of extracellular acidification. Addition of glucose to F. oxysporum var. lini cells also caused an increase in the cAMP level. No reliable increase could be demonstrated for the other sugars. Addition of proton ionophores such as DNP and CCCP at pH 5.0 caused both a large increase in the intracellular level of cAMP and in the activity of the plasma membrane H(+)-ATPase. Inhibition of the DNP-induced increase in the cAMP level by acridine orange also resulted in inhibition of the activation of plasma membrane H(+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Glucose-induced activation of the plasma membrane H(+)-ATPase in Fusarium oxysporum. 132 95

Anthracycline accumulation was evaluated by flow cytometry or radiolabeled drug assays in cells and cytoplasts (enucleated cells) prepared from parental and multidrug-resistant human K562 leukemia cells. Treatment with energy inhibitors, such as dinitrophenol (DNP) or sodium azide/deoxyglucose, led to a marked decrease in daunorubicin accumulation in parental cells and cytoplasts. Another ionophore, monensin, also caused a significant decrease in daunorubicin accumulation; however, ATPase inhibitors ouabain, vanadate, and N-ethylamaleimide had little or no effect. The lysosomatropic agents chloroquine and methylamine caused a moderate decrease in anthracycline accumulation. Fluorescence microscopy showed that the DNP-sensitive daunorubicin uptake occurred in a nonnuclear subcellular compartment. Studies using increasing daunorubicin concentrations demonstrated fluorescence quenching that occurred in the nonnuclear, DNP-sensitive compartment. The effect of inhibitors on the accumulation of rhodamine 123 and acridine orange strongly implicated lysosomes as the principal compartment of this inhibitable daunorubicin accumulation. Cytoplasts from P-glycoprotein containing multidrug-resistant K562 cells demonstrated a verapamil-reversible, decreased daunorubicin accumulation that was observed in resistant whole cells. Verapamil pretreatment of cytoplasts from resistant cells revealed the subcellular DNP-sensitive uptake present in parental cytoplasts. These studies demonstrate that cytoplasts are an effective means to study drug transport in mammalian cells without nuclear drug binding. Parental K562 cells and cytoplasts exhibit an energy-dependent accumulation of daunorubicin into cytoplasmic organelles that is also present in resistant cells and cytoplasts when P-glycoprotein mediated efflux is inhibited.
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PMID:Energy-dependent accumulation of daunorubicin into subcellular compartments of human leukemia cells and cytoplasts. 135 Feb 80

We characterized Mg(2+)-dependent ATPase activity in membranes from the renal cortex, the outer and inner stripes of the outer medulla, and papillary vesicles. In all regions, there was Mg(2+)-dependent ATPase activity that was resistant to oligomycin and vanadate and sensitive to N,N'-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide, and filipin. DCCD-Sensitive Mg(2+)-ATPase activity was highest in the inner stripe of the outer medulla and lowest in the cortex, with intermediate values in the outer stripe of the outer medulla and papilla. The Km for ATP, however, was similar among the different regions of the kidney. DCCD-Sensitive Mg(2+)-ATPase activity was critically dependent upon chloride with Km for Cl- in the range of 2-5 mM. In the presence of ATP, this ATPase was capable of H+ translocation, as assessed by acridine orange quenching. Inhibitors of ATPase activity prevented H+ translocation, which suggests that the Mg(2+)-ATPase represents, at least in part, an H(+)-ATPase. H+ transport was likewise critically dependent upon chloride, with similar Km. The effect of chloride on H+ translocation was blocked by the chloride channel inhibitor, diphenylamine-2 carboxylic acid. In the absence of chloride, H+ transport was abolished, but it could be partially restored by the creation of a favorable electric gradient by K+ and valinomycin. These studies demonstrate that the renal H(+)-ATPase exhibits different activities in various regions of the kidney. The ATPase activity and H+ translocation are critically dependent upon the presence of chloride, which suggests that chloride influences H+ translocation by dissipating the H+ gradient and acting at the catalytic site of the ATPase.
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PMID:ATP-dependent renal H+ translocation: regional localization, kinetic characteristics, and chloride dependence. 138 29

Intracellular acidic compartments serve several functions, including uptake of nutrients, processing and sorting of secreted and membrane-bound proteins, and even entry of viruses into cells. In this study, we examined the distribution of acidic compartments in normal human keratinocytes cultured in serum-free medium. Acridine orange was used to stain acidic organelles (red fluorescence), and adherent cells were evaluated by fluorescence microscopy and by interactive laser cytometry (ILC). Keratinocytes cultured in low [Ca++] (0.15 mM) exhibited morphologic characteristics associated with basal cells; red acidic vesicles in these cells were aggregated around the nucleus, sparing the peripheral cytoplasm. After 24 h of culture in high [Ca++] (1.5 mM) keratinocytes showed morphologic changes associated with differentiated cells, including increased number and dispersal of red vesicles to the periphery of the cytoplasm. Keratinocytes cultured in 0.15 mM [Ca++], but treated with phorbol 12-myristate 13-acetate (PMA, 5-100 ng/ml) to induce terminal differentiation, developed similar features. Incubation in media with either high [Ca++] or PMA also induced radial extension of the microtubule network, suggesting that the distribution of acidic organelles occurs along this network. Finally, crude keratinocyte membranes were evaluated by radioactive assay for the presence of three ion-translocating ATPase activities, plasma membrane Na/K ATPase, mitochondrial ATPase, and vacuolar H+ pump ATPase, the latter being the activity responsible for acidification of intracellular compartments. Both basaloid and differentiated keratinocytes exhibited similar vacuolar H+ pump ATPase activity, as measured by its sensitivity to bafilomycin.
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PMID:Increased number and microtubule-associated dispersal of acidic intracellular compartments accompany differentiation of cultured human keratinocytes. 153 43

The development of the Na/H antiporter was studied in renal brush border membrane vesicles (BBMV) from fetal and adult rabbits using isotopic and fluorescent techniques. The kinetics of the antiporter studied by 22Na+ uptake revealed that the Vmax was only 25% of that in the adult; however, the Km's for Na+ were not significantly different. These data were confirmed by a fluorescent assay using the pH-sensitive probe, acridine orange: the Vmax was significantly lower in the fetal BBMV. Conductive Na+ movement was estimated from amiloride-insensitive 22Na+ uptake and the rate of alkalinization induced by K+, an ion whose relative conductance was found to be similar to that of Na+. Although relative Na+ conductance was significantly greater in fetal BBMV, the lower Vmax in fetal vesicles could not be ascribed to this factor. Maternal administration of betamethasone (50 micrograms/kg intramuscularly) for 2 d before delivery significantly increased the Vmax of the antiporter to levels observed in the adult; Km was unaffected. Na/K ATPase activity increased fourfold after betamethasone, but the specific activities of four brush border marker enzymes and the kinetics of Na(+)-glucose cotransport were unchanged. These data indicate that there is a developmental increase in brush border Na/H exchange which is the result of an increase in the number and/or the turnover number of the carriers. Further, these data suggest that the postnatal increase in antiporter activity may be related to the surge in glucocorticoid concentration that occurs perinatally.
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PMID:Ontogeny of Na/H antiporter activity in rabbit renal brush border membrane vesicles. 164 51

SK&F 96067 [3-butyryl-4-(2-methylphenylamino)-8-methoxyquinoline] has been identified, from a novel class of 4-aminoquinolines, as a reversible inhibitor of the gastric (H+ + K+)-ATPase. This compound has been studied in gastric membrane vesicle preparations enriched in the (H+ + K+)-ATPase. At pH 7.0, SK&F 96067 inhibited K(+)-stimulated ATPase activity competitively with respect to the activating cation K+, with a Ki value of 0.39 +/- 0.05 microM. Under comparable conditions, SK&F 96067 was 32 times more potent as an inhibitor of the gastric (H+ + K+)-ATPase relative to the closely related (Na+ + K+)-ATPase. Studies in intact gastric vesicles showed that SK&F 96067 also inhibited hydrogen ion transport. Using the initial rate of acridine orange quenching as the index of acidification, an IC50 of 0.84 +/- 0.24 microM was observed. Steady state acidification, as measured by aminopyrine accumulation, was inhibited with greater potency (IC50 = 0.06 +/- 0.01 microM) consistent with the accumulation of this inhibitor into the intravesicular acidic space to a site of action on the inside (lumenal) face of the enzyme. Inhibition of ATPase activity in the presence of both SK&F 96067 and the K(+)-competitive (H+ + K+)-ATPase inhibitor, SCH 28080, indicated that their binding was mutually exclusive, consistent with SK&F 96067 acting at the same lumenal binding site as does SCH 28080. The steady-state inhibition kinetics of SK&F 96067 against K(+)-stimulated ATPase activity were followed as a function of pH. At pH 6.6 and 7.0 the inhibition was competitive with respect to the activating cation K+. At pH 7.5 and 8.1 a mixed pattern of inhibition was detected. Thus, at alkaline pH values, the binding of SK&F 96067 and K+ were no longer mutually exclusive. The potency of SK&F 96067 decreased as pH rose, consistent with the protonated form of the inhibitor being the preferred inhibitory species. A kinetic model is discussed, in which, at acidic pH, the protonated form of SK&F 96067 binds to the enzyme competitively with respect to K+, whereas, at alkaline pH, the neutral form of SK&F 96067 can bind simultaneously with K+.
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PMID:SK&F 96067 is a reversible, lumenally acting inhibitor of the gastric (H+ + K+)-ATPase. 164 19

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.
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PMID:Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients. 168 90

Langerhans cells (LC) are the principal antigen-presenting cells (APC) of squamous epithelia. We have previously shown that freshly isolated LC (fLC) are able to deliver endocytosed membrane MHC class II molecules into acidic environments, and that this capacity is lost when LC are placed in culture (cLC). Inasmuch as processing of antigens requires their passage through acidic compartments, we undertook the present study to examine the ability of fLC and cLC to take up acridine orange, and to identify proton-translocating ATPases in these cells. Using flow cytometry and fluorescence microscopy, acridine orange was observed to accumulate in acidic compartments in both fLC and cLC. Using a radioactive ATPase assay, crude membrane preparations from both fLC and cLC were shown to possess three types of ion-translocating ATPase, based on sensitivity to the following inhibitors: ouabain (Na+, K+ ATPase), oligomycin (mitochondrial F1F0 ATPase), and bafilomycin (vacuolar-type proton pump ATPase); the last type is responsible for acidification in vacuolar compartments. cLC displayed markedly less (less than 50%) total ATPase activity compared to fLC; however, the relative proportions of specific ATPases were similar in fLC and cLC. Combined use of the three inhibitors resulted in abrogation of only 25-40% of the total ATPase activity. Finally, treatment of LC with bafilomycin inhibited both acridine orange uptake and acidification of internalized HLA-DR molecules. These results confirm the ability of both fLC and cLC to acidify vacuolar compartments, thereby suggesting that lack of acidification of endocytosed membrane class II molecules in cultured cells is due to alternative routing to non-acidic organelles.
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PMID:Vacuolar acidification and bafilomycin-sensitive proton translocating ATPase in human epidermal Langerhans cells. 182 37

In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.
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PMID:A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane. 183 Dec 2

Bafilomycin A1 is known as a strong inhibitor of the vacuolar type H(+)-ATPase in vitro, whereas other type ATPases, e.g. F1,F0-ATPase, are not affected by this antibiotic (Bowman, E.M., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7972-7976). Effects of this inhibitor on lysosomes of living cultured cells were tested. The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when BNL CL.2 and A431 cells were treated with 0.1-1 microM bafilomycin A1. The effect was revealed by washing the cells. Both studies using 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and fluorescein isothiocyanate-dextran showed that the intralysomal pH of A431 cells increased from about 5.1-5.5 to about 6.3 in the presence of 1 microM bafilomycin A1. The pH increased gradually in about 50 min. In the presence of 1 microM bafilomycin A1, 125I-labeled epidermal growth factor (EGF) bound to the cell surface at 4 degrees C was internalized normally into the cells at 37 degrees C but was not degraded at all, in marked contrast to the rapid degradation of 125I-EGF in the control cells without the drug. Immunogold electron microscopy showed that EGF was transported into lysosomes irrespective of the addition of bafilomycin A1. These results suggest that the vacuolar type H(+)-ATPase plays a pivotal role in acidification and protein degradation in the lysosomes in vivo.
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PMID:Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. 183 76


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