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:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two modifications to Western blots which enhance immunochemical recognition have been developed. The first is transfer in carbonate buffer at pH 9.9, rather than the more commonly used Tris-glycine buffer at pH 8.3. This alteration improved the recognition of four of the five subunits of Escherichia coli F1-ATPase by monoclonal antibodies, the smaller subunits showing the greatest effects. Recognition of dinitrophenyl groups attached to the subunits by polyclonal antibodies was improved by the carbonate buffer only for the smallest ATPase subunit, epsilon. The second modification was incubation of the gel in mild buffers, designed to promote the renaturation of proteins, before the electrophoretic transfer step. The most effective buffer was 20% glycerol in 50 mM Tris-HCl, pH 7.4. Improvements in the signal obtained with monoclonal antibodies to all the subunits of ATPase were obtained by this procedure. As the subunits vary markedly in size, isoelectric point, and other properties, this method should be useful for most proteins. The fate of the 15,000-Da epsilon subunit, labeled with 125I, was followed through a blotting experiment. As long as no sodium dodecyl sulfate was added to the transfer buffer, epsilon was bound to nitrocellulose efficiently in either Tris-glycine or carbonate buffer. However, the epsilon was retained much more strongly during the subsequent incubation steps if the transfer was done in the carbonate buffer. The binding of epsilon to the nitrocellulose was even more stable when the gel had been treated with the buffered glycerol solution before transfer. These results indicate that the conditions under which epsilon subunit first encounters the nitrocellulose markedly affect the stability of binding during subsequent steps. The F1-ATPase was partially fragmented by treatment with proteases and then run on a gel and either transferred immediately in Tris-glycine buffer or else treated with the buffered glycerol solution and transferred in the carbonate buffer. The second blot gave stronger recognition of residual alpha subunit and fragments by an anti-alpha monoclonal antibody, with the largest improvement for the smaller fragments. This result suggests that the modified procedure may be particularly useful in enhancing the detection of small proteins.
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PMID:Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. 353 63

The effect of fluphenazine and related phenothiazine and thioxanthene derivatives on beef heart soluble mitochondrial ATPase (EC 3.6.1.3) was studied under a precise control of the Mg2+-ATP equilibrium. These drugs were shown to be reversible, noncompetitive inhibitors with respect to the substrate (the Mg . ATP complex). The inhibition was found to be dependent on the concentration of free magnesium ions, although free Mg2+ was not essential for the interaction of the inhibitors with the enzymatic protein. Bicarbonate anions, which are known to antagonize the effect of free Mg2+ on the enzyme kinetics, also antagonized the drug-induced inhibition. Concentrations giving 50% inhibition of enzyme activity were in the micromolar range. Inhibitory potencies increased when the pH of the reaction mixture was lowered from 8.2 to 6.9. Cleland [The Enzymes (P. D. Boyer, ed.), Vol. II. Academic Press, New York, 1--65 (1970)] analysis of the inhibition, by means of slope and intercept replots, indicated that the inhibition was the result of the interaction with more than one drug molecule. All drugs tested afforded complete protection against the cold-induced inactivation of soluble mitochondrial ATPase. These results point to a specific mode of inhibition that mimics, in some respects, the action of the natural inhibitor protein of mitochondrial ATPase.
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PMID:Magnesium-dependent inhibition of beef heart soluble mitochondrial adenosine triphosphatase by tricyclic antipsychotics. 612 79

Mg-ATPase of rat brain synaptic vesicles (SV) is considerably (by 85%) inhibited by dicyclohexyl carbodiimide (200 microM), a blocker of proton pumps, whereas orthovanadate (100 microM) does not produce any influence on the enzyme. Oligomycin (5 micrograms/ml) does not alter Mg-ATPase activity of the SV, whereas N-ethylmaleimide (300 microM) reduces it to a moderate degree, namely by 35%. This indicates that Mg-ATPase of the SV differs from mitochondrial ATPase. The protonophore p-trichloromethoxycarbonyl cyanide phenylhydrazone (20 microM) and bicarbonate anions (20 mM) stimulate slightly (by 12 to 25%) Mg-ATPase of the SV. Bicarbonate (20 mM) raises 1.8-2.1-fold Mg-ATPase activity of the mitochondria isolated from rat brain. It is assumed that the membrane of brain SV contains proton ATPase (H+-ATPase) differing from mitochondrial H+-ATPase in some of the properties.
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PMID:[Evidence for the presence of H+-ATPase in the membrane of brain synaptic vesicles in the rat]. 615 Jul 34

The membrane ATPase activities present in rat pancreas were studied to investigate the possible role of ATPase enzymes in HCO3(-) secretion in the pancreas. It was found that all the HCO3(-)-sensitive (anion-sensitive) ATPase activity was accountable as pancreatic mitochondrial ATPase, thus supporting the view that a distinct plasma membrane 'bicarbonate-ATPase' is not involved in HCO3(-) secretion in pancreas. A remarkably high Mg+- and CA2+-requiring ATPase activity (30 mumol ATP hydrolysed/min per mg) was found in the plasma membrane fraction (rho = 1.10-1.13). This activity has been characterized in some detail. It is inhibited by p-fluorosulfonylbenzoyladenosine, an affinity label analogue of ATP and the analogue appears to label covalently a protein of Mr approximately 35 000. The (Ca2+ + Mg2+)-ATPase activity did not form a 'phosphorylated-intermediate' and was vanadate-insensitive. These and other tests have served to demonstrate that the (Ca2+ + Mg2+)-ATPase activity is different in properties from (Na+ + K+)-ATPase, Ca2+-ATPase, (H+ + K+)-ATPase or mitochondrial H+-ATPase. Apart from the (Ca2+ + Mg2+)-ATPase of plasma membrane and mitochondrial ATPase, the only other membrane ATPase activities noted were (Na+ + K+)-ATPase, which occurred in the same fractions as the (Ca2+ + Mg2+)-AtPase at rho = 1.10-1.13 and was of surprisingly low activity, and an ATPase activity in light membrane fractions (rho - 1.08-1.09) derived from zymogen granule membranes. At this time, therefore, there is no obvious candidate for an ATPase activity at the luminal surface of pancreatic cells which is directly involved in ion transport, but the results presented here direct attention to the high activity (Ca2+ + Mg2+)-ATPase in the plasma membrane fraction.
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PMID:Membrane adenosine triphosphatase activities in rat pancreas. 625 65

The results of studies on the initial velocity in hydrolysis reactions of ATP and other nucleoside triphosphates with beef liver mitochondrial ATPase can be summarized as follows. 1. Double reciprocal plots of substrate concentration vs. initial velocity were linear for all the nucleoside triphosphates tested except for ATP, which showed a negative cooperativity. 2. Bicarbonate ion increased the rate of ATP hydrolysis and diminished its negative cooperativity, whereas hydrolysis of other nucleoside triphosphates was only slightly affected by the anion. 3. An excess of nucleoside triphosphate apparently inhibited its own hydrolysis for all kinds of nucleoside triphosphates tested, whereas an excess of magnesium ion apparently inhibited only ATP hydrolysis. 4. Inhibition of ATPase activity with an excess of magnesium ion was no longer observed when the reaction was carried out at low temperature (10 degrees C) or in the presence of sulfate. Under these conditions, the kinetics of ATP hydrolysis were apparently of simple Michaelis-Menten type. These observations suggest the existence of two states ("A" and "N") of beef liver mitochondrial ATPase. The state "A" is characterized by phenomena specifically affecting ATP hydrolysis, such as the inhibition by excess magnesium ion, and the negatively cooperative profile in the dose-response curve of ATP. In the state "N" proceed the hydrolysis reactions of other nucleoside triphosphates and of ATP under limited conditions. The two states ("A" and "N") can be related to an enzyme model with a catalytic site and a regulatory site. Computer stimulation revealed that such a model could account well for the experimental data.
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PMID:Implications of the existence of two states of beef liver mitochondrial adenosine triphosphatase as revealed by kinetic studies. 645 83

Flow cytometric cell-by-cell evaluation of NH4Cl acidification of human Chang cells showed that at steady state, 3% of the cells remained alkalinized (> pHi 7) over an extended period (up to 80 min) despite the absence of extracellular Na+ and HCO3-. In fluorescence microscopy, the acidification-resistant cells were characteristically rounded M-phase cells. Both mean cytosolic pH and M-phase alkalinity were however sensitive to (a) azide and oligomycin, inhibitors of F-ATPase (ATP synthase), and to (b) vanadium ions, the phosphate analogue of P-ATPase (ATP-hydrolyzing), in dose-dependent and time-dependent manners. Dead cell indices were constant at approximately 10%. Thiocyanate chaotrophic anions, which cleave the V-ATPase structure, had no effect. Since ATP synthesizing F-ATPase (ATP synthase) is coupled to ATP-hydrolyzing P-ATPase as 'master-&-slave', azide- and oligomycin-sensitivity corroborated with vanadate-sensitivity in suggesting energized proton pumping modulating (a) M-phase alkalinity and (b) cytosolic pH, against acidification.
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PMID:Azide- and vanadate-sensitive M-phase alkalinity and cytosolic acidification of Chang liver cells. 808 35

Pneumocystis carinii is an opportunistic fungus which causes interstitial pneumonia in patients with acquired immunodeficiency syndrome (AIDS). Cytoplasmic pH (pHi) regulation in short-term-cultured P. carinii trophozoites was studied using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein. With an extracellular pH of 7.4, the mean baseline pHi of P. carinii trophozoites was 7.40 +/- 0.10 (n = 8). This steady-state pHi was not significantly affected in the absence of extracellular Na+ or K+. Moreover, steady-state pHi was maintained in the nominal absence of HCO3- and was not affected by the Cl-/HCO(3-)-exchanger inhibitor 4, 4'-di-isothiocyanato-dihydrostilbene-2, 2'-disulphonic acid (100 microM), or the Na+/H(+)-exchanger inhibitor N-ethyl-N-isopropylamiloride (100 microM). In contrast, the general inhibitors of ATPases, N-ethylmaleimide (1 mM), and dicyclohexylcarbodi-imide (100 microM), and the inhibitor of yeast H(+)-ATPase, diethylstilbestrol (12.5-100 microM), decreased pHi, while the K+/H(+)-ATPase inhibitor omeprazole (50-400 microM), and the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (1-5 microM) only produced a dose-dependent acidification of the cells when used at high concentrations. In addition, steady-state pHi depended on the availability of cellular ATP, since it was decreased by the ATP synthase inhibitors oligomycin (1 microgram/ml) and sodium azide (1 mM), and by the uncoupler of oxidative phosphorylation carbonyl cyanide p-trifluorophenylhydrazone (1 microM), agents that were able to deplete significantly the intracellular ATP levels. Taken together, these results are consistent with an important role of an H(+)-ATPase similar to those found in other fungi in the regulation of pHi homoeostasis in P. carinii trophozoites.
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PMID:An H(+)-ATPase regulates cytoplasmic pH in Pneumocystis carinii trophozoites. 868 17

We have previously isolated the yeast nuclear gene OXA1 and showed that Oxa1p is required for the formation of the cytochrome c oxidase and ATP synthase complexes. We have expressed Oxa1p in E. coli and shown that it is toxic and rapidly degraded. Nevertheless, a truncated protein was successfully expressed and antibodies have been raised against this truncated protein. These antibodies recognise a protein in mitochondrially enriched fractions. In vitro mitochondrial import experiments demonstrate that the import of Oxa1p is accompanied by the cleavage of a long pre-sequence. Osmotic swelling and alkaline carbonate extraction show that Oxa1p is an integral membrane protein located in the inner membrane of mitochondria. The relationships between the sub-mitochondrial location and the function of Oxa1p are discussed.
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PMID:Oxa1p, which is required for cytochrome c oxidase and ATP synthase complex formation, is embedded in the mitochondrial inner membrane. 910 37

Oxa1p is a mitochondrial inner membrane protein that is mainly required for the insertion/assembly of complex IV and ATP synthase and is functionally conserved in yeasts, humans, and plants. We have isolated several independent suppressors that compensate for the absence of Oxa1p. Molecular cloning and sequencing reveal that the suppressor mutations (CYT1-1 to -6) correspond to amino acid substitutions that are all located in the membrane anchor of cytochrome c1 and decrease the hydrophobicity of this anchor. Cytochrome c1 is a catalytic subunit of complex III, but the CYT1-1 mutation does not seem to affect the electron transfer activity. The double-mutant cyt1-1,164, which has a drastically reduced electron transfer activity, still retains the suppressor activity. Altogether, these results suggest that the suppressor function of cytochrome c1 is independent of its electron transfer activity. In addition to the membrane-bound cytochrome c1, carbonate-extractable forms accumulate in all the suppressor strains. We propose that these carbonate-extractable forms of cytochrome c1 are responsible for the suppressor function by preventing the degradation of the respiratory complex subunits that occur in the absence of Oxa1p.
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PMID:Mutations in the membrane anchor of yeast cytochrome c1 compensate for the absence of Oxa1p and generate carbonate-extractable forms of cytochrome c1. 975 93

When Zostera marina was irradiated after a period of darkness, initiation of photosynthetic O2 evolution occurred in two phases. During a lag phase, lasting 4 to 5 min, photosynthesis was supported by a diffusive entry of CO2. Photosynthesis then rapidly increased to its full rate. Tris buffer, at a concentration of 50 mm, completely inhibited this increase without affecting CO2-supported photosynthesis during the lag phase. These results verify that the increase in photosynthesis after the lag phase depended on an activation of bicarbonate (HCO3-) utilization through acid zones generated by proton pumps located to the outer cell membrane. In similar experiments, 6.25 microm of the mitochondrial ATPase blocker oligomycin inhibited photosynthetic HCO3(-) utilization by more than 60%. Antimycin A, a selective blocker of mitochondrial electron transport, caused a similar inhibition of HCO3(-) utilization. Measurements at elevated CO2 concentrations verified that neither oligomycin nor antimycin interfered with linear photosynthetic electron transport or with CO2 fixation. Thus, a major part of the ATP used for the generation of acid zones involved in HCO3(-) utilization in Z. marina was derived from mitochondrial respiration.
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PMID:Photosynthetic utilization of bicarbonate in Zostera marina is reduced by inhibitors of mitochondrial ATPase and electron transport. 1843 9


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