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)

Dicyclohexylcarbodiimide-resistant mutants of Escherichia coli were isolated and characterized In one mutant the unc genes and affects the membrane-integrated part of the ATP synthetase. The sensitivity of ATP synthetase functions to N,N' -dicyclohexylcarbodiimide was compared in wild-type and mutant membranes. The membrane-integrated part of the wild-type ATP synthetase is highly sensitive to ATP-dependent membrane energization and restoration of lactate-dependent energization of ATPase-depleted membranes. In mutant membranes this concentration has only a slight effect on these activities whereas a severe inhibition is obtained at 200 muM. Using the highly water-soluble 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide theactivities of wild-type and mutant membranes are inhibited to the same extent. TheATP synthetase of wild-type and mutant was partially purified and incorporated muM. Uinto liposomes. These showed an uncoupler-sensitive ATP-32Pi exchange and ATP-dependent quenching of acridine-dye fluorescence. The activities of mutant and wild-type proteoliposomes exhibit the same pattern of sensitivity to dicyclohexylcarbodiimide as the corresponding membranes.
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PMID:A mutant ATP synthetase of Escherichia coli with an altered sensitivity to N,N' -dicyclohexylcarbodiimide: characterization in native membranes and reconstituted proteoliposomes. 1 31

Fluorescent amines, 9-aminoacridine, acridine orange and quinacrine, were used as probes for a pH gradient (deltapH) across gastric microsomal vesicles. Analysis of probe uptake data indicates that 9-aminoacridine distributes across the membrane as a weak base in accordance with the deltapH. On the other hand, acridine orange and quinacrine show characteristics of binding to membrane sites in addition to the accumulation in response to deltapH. A discussion of the advantages and limitations of the probes is presented. Application of these probes to pig gastric microsomal vesicles indicates that that K+-stimulated ATPase is responsible for the transport of H+ into the vesicles and thus develops a deltapH across the membrane. The deltapH generated by the K+-ATPase has a definite requirement for internal K+. The proton gradient can be discharged slowly after ATP depletion or rapidly either by detergent disruption of the vesicles or by increasing their leakiness using both H+ and K+ ionophores. On the other hand, the sole use of the K+ ionophore, valinomycin, stimulates the ATP-induced formation of deltapH by increasing the availability of K+ to internal sites. This stimulation by valinomycin requires the presence of permeable anions like Cl-. Analysis of the Cl- requirement indicates that in the presence of valinomycin the net effect is the accumulation of HCl inside the gastric vesicles. With an external pH of 7.0, the ATP-generated deltapH was calculated to be from 4 to 4.5 pH units. The results are consistent with the hypothesis that the K+-stimulated ATPase drives a K+/H+ exchange across the gastric vesicles. Since other lines of evidence suggest that these gastric microsomes are derived from the tubulovesicular system of the oxyntic cell, the participation of the ATP-driven transport processes in gastric HCl secretion is of interest.
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PMID:A study of H+ transport in gastric microsomal vesicles using fluorescent probes. 2 82

The Mg2+-dependent, K+-stimulated ATPase of microsomes from pig gastric mucosa has been studied in relation to observed active H+ transport into vesicular space. Uptake of fluorescent dyes (acridine orange and 9-aminoacridine) was used to monitor the generated pH gradient. Freeze-fracture electron microscopy showed that the vesicular gastric microsomes have an asymmetric distribution of intramembraneous particles (P-face was particulate; E-face was relatively smooth. Valinomycin stimulated both dye uptake and K+-ATPase (valinomycin-stimulated K+-ATPase); stimulation by valinomycin was due to increased K+ entry to some intravesicular activating site, which in turn depends upon the accompanying anion. Using the valinomycin-stimulated K+-ATPase and H+ accumulation as an index, the sequence for anion permeation was NO-3 greater than Br- greater than Cl- greater than I- greater than acetate approximately isethionate. When permeability to both K+ and H+ was increased (e.g using valinomycin plus a protonophore or nigericin), stimulation of K+-ATPase was much less dependent on the anion and the observed dissipation of the vesicular pH gradient was consistent with an 'uncoupling' of ATP hydrolysis from H+ accumulation. Thiocyanate interacts with valinomycin inhibiting the typical action of the K+ ionophore. But stimulation of ATPase activity was seen by adding 10 mM SCN- to membranes preincubated with valinomycin. From the relative activation of the valinomycin-stimulated K+-ATPase, it appears that SCN- is a very permeant anion which can be placed before NO-3 in the sequence of permeation. Valinomycin-stimulated ATPase and H+ uptake showed similar dependent correlations, including: dependence on [ATP] and [K+], pH optima, temperature activation, and selective inhibition by SH- or NH2-group reagents. These results are consistent with a pump-leak model for the gastric microsomal K+-ATPase which was simulated using Nernst-Planck conditions for passive pathways and simple kinetics for the pump. The pump is a K+/H+ exchange pump requiring K+ at an internal site. Rate of K+ entry would depend on permeability to K+ as well as the counterion, either (1) the anion to accompany K+ or (2) the H+ efflux path as an exchange ion. The former leads to net accumulation of H+ and anion, while the latter results in non-productive stimulation of ATP hydrolysis.
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PMID:Potassium-stimulated ATPase activity and hydrogen transport in gastric microsomal vesicles. 3 10

Transport activity of the hog gastric (H+ + K+)-ATPase system was measured either as the formation of proton gradient using the dye probe acridine orange or as the formation of a proton diffusion potential using the cyanine dye 3,3'-diethyloxdicarbocyanine iodide in the presence of the protonophore tetrachlorosalicylanilide. The development of these gradients has been compared in K+ media in the presence of either Cl- or SO4-2 as the anionic species. This comparison of proton diffusion potential formation to proton gradient formation has been used to demonstrate that a Cl- conductance in this vesicular system results from limited enzymic digestion with either trypsin or alpha-chymotrypsin from the ageing process itself. The possible significance of this finding is discussed.
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PMID:Induction of a chloride conductance in gastric vesicles by limited trypsin or chymotrypsin digestion or ageing. 3 2

Ethidium bromide, in addition to combination with mitochondrial nucleic acids, is a phosphorylation inhibitor during glutamate and succinate respiration by mitochondria. Exhaustive washing of ethidium bromide-treated mitochondria did not relieve the inhibition nor significantly decrease the amount of bound dye. Dialysis against a cation exchange resin at 3 degrees for 17 hr removed about 97% of bound dye. This restored phosphorylating capacity to that of untreated mitochondria which had also been dialyzed against the resin. Since state 3 respiration was diminished and state 4 was unaffected by the presence of the acridine dye, and since neither swelling of mitochondria nor release of latent ATPase was observed, then ethidium bromide was not an electron transport inhibitor nor an uncoupler of oxidative phosphorylation. Inhibition of metabolic processes by ethidium bromide may be due in part to depressed generation of mitochondrial ATP.
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PMID:Ethidium bromide inhibits mitochondrial phosphorylating oxidation. 12 52

Oxidative phosphorylation, ATP-32Pi exchange, ATP-dependent quenching of acridine-dye fluorescence, ATP-dependent transhydrogenase and ATP-dependent transport of thiomethyl beta-D-galactoside are shown to be experimentally equivalent tools to study the functional state of the ATPase complex in Escherichia coli wild-type and mutant strains defective in oxidative phosphorylation. According to these criteria ten mutants in the ATPase complex were classified having lesions in the unc A,B region of the chromosome. The first mutant type lacks ATPase activity, but the membrane-integrated part of the complex remains functional (class I). The second mutant type lacks a functional membrane-integrated part, but retains ATPase activity (class II). The third mutant type is shown to be defective in both parts of the ATPase complex (class III).
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PMID:The use of several energy-coupling reactions in characterizing mutants of Escherichia coli K12 defective in oxidative phosphorylation. 13 25

ATP-induced transport by fractions of frog gastric microsomes prepared either by density gradient centrifugation of by free flow electrophoresis were K+ dependent and hence considered due to a K+-activated ATPase. Significant activity of this enzyme was, however, only found in the anodic peak of the free flow electrophoretic separation, which in addition to separating transporting from non-transporting particles, also separated membranes containing a phosphorylatable peptide (Mr=105 000) region as the major peptide on SDS-polyacrylamide gel electrophoresis from those containing a peptide (Mr=44 000) on SDS-polyacrylamide gel electrophoresis. H+ uptake, measured either by acridine orange or 3,3'-diethyloxadicarbocyanine + tetrachlorosalicylanilide absorbance changes was dependent on K+ intravesicularly. Using 86Rb+, active extrusion of the cation followed ATP addition. SCN-, an inhibitor of acid secretion did not affect the latter, but blocked signals due to H+ uptake, in contrast to mammalian preparations.
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PMID:Transport characteristics of frog gastric membranes. 15 47

A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H(+)-pump, passive H+ conductance, and Na+/H+ exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H+ transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. As measured by potential-driven H+ accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15-30%, Na+/H+ exchange by 20-55%, and H+ conductance was unchanged. Osmotic water permeability (Pf) was 570 microns/s and was inhibited 85-90% by 0.6 mM HgCl2; proteinase treatment did not affect Pf or the HgCl2 inhibition. In EV, NEM-sensitive H+ accumulation and ATPase activity were inhibited by greater than 95%. Pf (140 microns/s) and HgCl2 inhibition (75-85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na+/H+ exchange by proteinase digestion. The lack of effect of proteinases on water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H(+)-ATPase and Na+/H+ exchanger, has no functionally important membrane domains that are sensitive to proteolysis.
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PMID:Proteinases inhibit H(+)-ATPase and Na+/H+ exchange but not water transport in apical and endosomal membranes from rat proximal tubule. 130 58

Trans cisternal elements of the Golgi apparatus from rat liver, identified by thiamin pyrophosphatase cytochemistry, were isolated by preparative free-flow electrophoresis and were found to undergo acidification as measured by a spectral shift in the absorbance of acridine orange. Acidification was supported not only by adenosine triphosphate (ATP) but nearly to the same degree by inorganic pyrophosphate (PPi). The proton gradients generated by either ATP or PPi were collapsed by addition of a neutral H+/K+ exchanger, nigericin, or the protonophore, carbonyl cyanide m-chlorophenylhydrazone, both at 1.5 microM. Both ATP hydrolysis and ATP-driven proton translocation as well as pyrophosphate hydrolysis and pyrophosphate-driven acidification were stimulated by chloride ions. However, ATP-dependent activities were optimum at pH 6.6, whereas pyrophosphate-dependent activities were optimum at pH 7.6. The Mg2+ optima also were different, being 0.5 mM with ATP and 5 mM with pyrophosphate. With both ATPase and especially pyrophosphatase activity, both by cytochemistry and analysis of free-flow electrophoresis fractions, hydrolysis was more evenly distributed across the Golgi apparatus stack than was either ATP- or PPi-induced inward transport of protons. Proton transport colocalized more closely with thiamin pyrophosphatase activity than did either pyrophosphatase or ATPase activity. ATP- and pyrophosphatase-dependent acidification were maximal in different electrophoretic fractions consistent with the operation of two distinct proton translocation activities, one driven by ATP and one driven by pyrophosphate.
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PMID:Pyrophosphate-induced acidification of trans cisternal elements of rat liver Golgi apparatus. 131 63

Proton transport by the vanadate-sensitive ATPase in plasma membrane (PM) vesicles from the marine unicellular microalga Platymonas viridis was investigated. The ATP-dependent generation of delta pH across the membranes of PM vesicles was followed by the changes in the absorbance of the aminoacridine probe, Acridine orange. Na+ caused the decay of delta pH generated by the ATPase, the rate of the decay being dependent on the concentrations of Na+ added. The phenomenon was specific for Na+. Amiloride inhibited Na(+)-dependent delta pH decay. The experiments support the idea of a Na(+)-extruding mechanism (H(+)-translocating ATPase plus Na+/H+ antiporter) operating in the PM of marine alga Pl. viridis.
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PMID:H(+)-translocating ATPase and Na+/H+ antiport activities in the plasma membrane of the marine alga Platymonas viridis. 132 76


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