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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)

1. Preincubation of MgATP submitochondrial particles with EDTA or Tris.HCl liberated a measurable amount of ATPase inhibitor that could be rapidly purified using only trichloroacetic acid precipitation and heat treatment. 2. In spite of the emergence of high ATPase activity, a considerable amount of ATPase inhibitor was left in the particles. Comparative analysis of other submitochondrial preparations indicated that only AS-particles were effectively depleted. 3. The high ATPase activity of inhibitor-deficient particles, was labile at low temperature provided that the exposure to cold was done in the presence of MgATP. Other nucleotides could not substitute for ATP. Glycerol inhibited and salts enhanced the cold inactivation of membrane-bound F1-ATPase. Isolation of F1-ATPase from cold-inactivated particles yielded a soluble preparation of correspondingly lower activity. 4. It is concluded that together with the increase of ATPase activity, the ATP-dependent cold lability of membrane-bound F1-ATPase and the dislocation of ATPase inhibitor at non operative sites reveal the extent of ATPase complex disorganization.
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PMID:Cold lability of membrane-bound F1-ATPase. 14 42

The gramicidin channel contains a single strand of water molecules associated through hydrogen bonds. Previous work has shown that channels of similar size are formed by association of transmembrane alpha helices of synthetic leucine-serine peptides. Both types of channels translocate protons with considerable selectivity relative to other cations, and it has been proposed that the selectivity arises by proton "hopping" along hydrogen-bonded chains of water, whereas other cations must cross by ordinary diffusion processes. It is possible that a similar mechanism underlies proton transport in the Fo subunit of the F1F0 ATP synthase. Using the gramicidin channel as a model, we have tested whether a single strand of water is kinetically competent to translocate protons at a rate sufficient to support known rates of ATP synthesis. We found that the gramicidin channel saturates at approximately 530 pS of protonic current in 4 M HCl, more than sufficient for typical ATP synthesis rates. It follows that proton diffusion to a putative channel in Fo, rather than the channel itself, may limit ATP synthesis rates.
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PMID:Proton conductance by the gramicidin water wire. Model for proton conductance in the F1F0 ATPases? 171 64

This report describes the first isolation and molecular characterization of the mitochondrial F1-ATPase from Trypanosoma brucei. The isolation procedure utilized is a modified chloroform extraction procedure. In contrast to earlier reports on the F1-ATPase from other trypanosomatids, the F1-ATPase we have isolated from the procyclic form of T. brucei a complex composed of five distinct subunits. Apparent molecular weights of these subunits are 55,000 [alpha], 42,000 [beta], 32,000 [gamma], 22,000 [delta], and 17,000 [epsilon]. The F1 moiety which possesses the active site of the H(+)-ATPase has an ATPase activity in the standard Tris-HCl coupled enzyme assay with a Vmax of 22.96 mumol min-1 (mg protein)-1 and a Km value of 0.60 mM. This ATPase activity is cold labile and is not susceptible to oligomycin inhibition as is the membrane bound enzyme. Upon reconstitution with F1-ATPase depleted membranes (urea particles) the ATPase regains oligomycin sensitivity to the same extent as that found in the intact inner membrane vesicles. ATP synthesis is also restored to these particles upon reconstitution with F1. These results indicate that this F1-ATPase as isolated is intact with respect to all the critical H(+)-ATPase functions.
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PMID:The mitochondrial ATP synthase of Trypanosoma brucei: isolation and characterization of the intact F1 moiety. 214 43

Ion-selective electrodes recorded the pH (7.49 +/- 0.05, n = 8) and pCa (6.72 +/- 0.03, n = 40) in samples (approximately 1 microliter) of isolated Myxicola axoplasm mounted within 760-micron diameter plastic tubes. We determined the interactions between Ca2+ and H+ on axoplasmic buffers by microinjecting CaCl2 or HCl into the axoplasmic samples at a distance 75-125 micron from the tips of the electrodes (distance = r). When axoplasmic pH was lowered 0.97 +/- 0.095 from its resting value (measured at r = 125 micron) by injecting 4 nmol HCl, pCa dropped 0.30 +/- 0.05 (n = 6). When expressed in units of concentration, these data show that a HCl injection of approximately 4 mmol/l axoplasm increased H+ and Ca2+ activity by approximately 0.3 microM. Lowering axoplasmic pCa 2.20 +/- 0.43 (r = 75 micron) (n = 3) by injecting 40 pmol CaCl2 had only a small effect on pH. In other experiments, two Ca2+ electrodes measured the Ca2+ activity 125 and 375 micron from the site of CaCl2 injection. Evidence of Ca2+ buffering was obtained when the Ca2+ activity at these two locations was below that expected for simple Ca2+ diffusion away from the injection site. Centrifuged axoplasm (100,000 g) taken from the bottom of the centrifuge tube had a somewhat greater Ca2+ buffering capacity than that taken from the top of the tube. Electron microscopic studies of the centrifuged axoplasm showed a greater concentration of mitochondria and other axoplasmic vesicles in the bottom of the centrifuge tube. Ruthenium red (20-40 micrograms/ml) greatly reduced Ca2+ buffering. The mitochondrial inhibitors CN (2 mM) and oligomycin (a mixture of oligomycin A, B, and C, 5 micrograms/ml) also reduced Ca2+ buffering but were not as effective as ruthenium red. Axoplasm in which ATP and mitochondrial substrates were removed by dialysis was unable to lower free Ca2+ when the concentration of this ion was elevated to approximately 10 microM. In the presence of oligomycin to block mitochondrial ATPase, and with Mg2+ -ATP as the only source of energy, axoplasm lowered Ca2+ activity slowly; with succinate as the only metabolic substrate, axoplasm rapidly lowered the Ca2+ activity from approximately 10 microM to below 1 microM.
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PMID:Calcium and proton buffering and diffusion in isolated cytoplasm from Myxicola axons. 242 Jan 93

The F0 portion of the rat liver mitochondrial ATP synthase (F0F1-ATPase) has been purified by a rapid, high yield procedure. F0 is selectively extracted from inner membrane vesicles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) after prior treatment of the vesicles with guanidine HCl to remove F1. The resultant F0 is functional in proton translocation assays and separates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis into four major and three minor Coomassie-stainable bands, all with apparent molecular masses below 30 kDa. This CHAPS-purified F0 preparation was characterized in detail for its capacity to interact with the unique probe diethylstilbestrol (DES) which, depending on conditions, has been shown to interact with rat liver F0F1 to either inhibit or promote ATP hydrolysis (McEnery, M. W., and Pedersen, P.L. (1986) J. Biol. Chem. 261, 1745-1752). DES-inhibitory sensitivity could be conferred on F1-ATPase activity with the same concentration dependence on F0 as conferral of oligomycin sensitivity. DES was shown also to inhibit the magnitude of valinomycin induced proton influx, while initiating proton efflux in asolectin vesicles reconstituted with F0 and loaded with K+. The potency of DES in producing the latter effects was shown to be highly dependent on hydroxyl groups in "para" positions of the two benzene rings within the DES molecule. Finally, in the absence of F0, DES was shown to act as a catalyst of proton influx in K+-loaded asolectin vesicles upon addition of valinomycin. A model based on the structure of DES is presented to account for both the inhibitory and uncoupling properties of this compound.
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PMID:F0 "proton channel" of rat liver mitochondria. Rapid purification of a functional complex and a study of its interaction with the unique probe diethylstilbestrol. 254 97

The modification of both beta-Tyr-368 and beta-His-427 can be correlated with the loss of activity observed when the bovine mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoyl[3H]adenosine ([3H]FSBA). At pH 8.0, where the rate of inactivation is fast, beta-Tyr-368 is modified predominantly, while at pH 6.0, where the rate of inactivation is slow, beta-His-427 is modified predominantly. At pH 7.0, the 2 residues are modified with about equal efficiency. When the F1-ATPase was inactivated by 80% at pH 6.5, 7.0, and 7.5, the sum of radioactivity incorporated into beta-Tyr-368 and beta-His-427 was 1.99, 1.87, and 1.82 mol of label incorporated per mol of enzyme, respectively. Examination of the rate of inactivation of the enzyme by FSBA as a function of pH revealed two pKa values, one of about 7.6 associated with the modification of beta-Tyr-368 and the other of about 5.8 associated with the modification of beta-His-427. The inactivation of the F1-ATPase by FSBA exhibited an initial fast rate followed by a slower rate in triethanolamine-HCl, pH 7.0. In contrast, only a single rate, equivalent to the fast phase of inactivation in the absence of phosphate, was observed in 0.2 M phosphate, pH 7.0. The dependence of this stimulation on phosphate concentration is sigmoidal with half-maximal stimulation occurring at approximately 160 mM. The ratio of 3H incorporated into beta-Tyr-368 to that incorporated into beta-His-427 was approximately the same during the fast and slow phases of inactivation in triethanolamine-HCl, pH 7.0. Approximately the same ratio was observed when the enzyme was modified during the single phase of inactivation exhibited in the presence of 0.2 M phosphate, pH 7.0. The sum of the 3H incorporated into beta-Tyr-368 and beta-His-427 during inactivation of the F1-ATPase from bovine heart mitochondria by [3H]FSBA in the presence and absence of phosphate was linear and extrapolated to a value of about 2.6 residues modified on complete inactivation of the enzyme. From these data, it is concluded that FSBA binds to a single binding site on the beta subunits of the enzyme where it reacts with either beta-Tyr-368 or beta-His-427 in mutually exclusive reactions. All three beta subunits must be modified in this manner for complete inactivation to be observed.
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PMID:Three copies of the beta subunit must be modified to achieve complete inactivation of the bovine mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoyladenosine. 287 Oct 17

The rates of hydrolysis of acetyl phosphate in the presence of 0.1 M NaOH and of ATP in the presence of either 1 M HCl or 1 M NaOH were measured at different temperatures and in the presence of different concentrations of the organic solvents dimethyl sulfoxide or ethylene glycol. Under all conditions tested, there was a progressive increase in the rate constant of hydrolysis of both phosphate compounds as the water activity of the medium was decreased by the addition of organic solvents. At 25 degrees C, substitution of 70% of the water of the medium by dimethyl sulfoxide promoted an increase of two orders of magnitude in the rate constant of acetyl phosphate hydrolysis. In the presence of 80% and 90% dimethyl sulfoxide the rate of acetyl phosphate hydrolysis increased by more than two orders of magnitude and was so fast that it could not be measured with the method used. The effect of organic solvents on the rate of ATP hydrolysis was less pronounced than that observed for acetyl phosphate hydrolysis. At 30 degrees C, substitution of 90% of water by an organic solvent promoted a 4-6-fold increase of the rate of ATP hydrolysis. Acceleration of either acetyl phosphate or ATP hydrolysis rates was promoted by a decrease in both activation energies (Ea) and in entropies of activation delta S. The data obtained are discussed with reference to the mechanism of catalysis of enzymes involved in energy transduction such as the Ca2+-ATPase of sarcoplasmic reticulum and the F1-ATPase of mitochondria.
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PMID:Role of water activity on the rates of acetyl phosphate and ATP hydrolysis. 289 8

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

We found that mitochondrial DNA (mtDNA) isolated from Physarum polycephalum fragmented itself in weak ionic solutions. The mtDNA was dissolved in STE (saline Tris-EDTA: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), TE (10 mM Tris-HCl, 1 mM EDTA) and DW, and then electrophoresed in an agarose gel. The intact 86-kbp mtDNA band was seen in STE, but several novel bands appeared in TE and DW. In TE, two discrete bands appeared at 6.7-kbp (alpha-band) and 5.0-kbp (beta-band), whereas at least 17 discrete bands were observed in distilled water (DW). These fragmentation patterns were not stoichiometric, as seen when using restriction endonucleases, but were clearly different from the degradation of DNA caused by a physical shearing force or a contaminating nuclease. In this paper, we characterize this in vitro fragmentation of mtDNA from P. polycephalum. We located 19 fragments, including the alpha and beta fragments, on a mtDNA restriction map, and demonstrated that these cleavage sites were S1 nuclease-sensitive regions, which are single-stranded DNA regions such as nicks and gaps in the mtDNA. The alpha and beta fragments are derived from the region encoding ribosomal RNAs (rRNAs) and the ATP synthase (atpA) gene, while the other 17 fragments are not derived from any specific region, but the cleavage sites are located throughout the mtDNA molecule. In P. polycephalum, it is well known that the growth rate of macroplasmodia decreases with aging. Equal amounts of mtDNA from juvenile and aged macroplasmodia were electrophoresed and the frequency of the beta fragment in each sample was measured. The ratio of the beta band to the total signal including background was estimated to be 3.3-4.0% in juvenile macroplasmodia, whereas it increased to 8.3-28.2% in aged macroplasmodia. This result suggests that the in vitro fragmentation of mtDNA is associated with macroplasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age.
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PMID:In vitro DNA fragmentation of mitochondrial DNA caused by single-stranded breakage related to macroplasmodial senescence of the true slime mold, Physarum polycephalum. 1074 69

We have developed a mathematical model in concert with an assay that allows us to calculate proton (H+) flux and conductance through a single FO of the F1FO ATP synthase. Lipid vesicles reconstituted with just a few functional FO from Escherichia coli were loaded with 250 mM K+ and suspended in a low K+ solution. The pH of the weakly buffered external solution was recorded during sequential treatment with the potassium ionophore valinomycin, the protonophore carbonyl cyanide 3-chlorophenylhydrazone, and HCl. From these pH traces and separate determinations of vesicle size and lipid concentration we calculate the proton conductance through a single FO sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wild-type FO has a proton flux of 3100 +/- 500 H+/s/FO at a transmembrane potential of 106 mV (25 degrees C and pH 6.8). This corresponds to a proton conductance of 4.4 fS.
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PMID:Determination of proton flux and conductance at pH 6.8 through single FO sectors from Escherichia coli. 1533 19


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