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)

The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1 (ECF1) has been found to be ligand-dependent, as measured indirectly by the activation of the enzyme that occurs on protease digestion, or when followed directly by monitoring the cleavage of this subunit using monoclonal antibodies. The cleavage of the epsilon subunit was fast in the presence of ADP alone, ADP + MG2+, ATP + EDTA, or AMP-PNP, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site(s). The half-maximal concentration of Pi required in the presence of ADP + Mg2+ to protect the epsilon subunit from cleavage by trypsin was 50 microM, which is in the range measured for the high-affinity binding of Pi to F1. The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + Mg2+ + Pi, the epsilon subunit cross-linked to beta in high yield. With ATP + EDTA or ADP + Mg2+ (no Pi), the yield of the beta-epsilon cross-linked product was much reduced. We conclude that the epsilon subunit undergoes a conformational change dependent on the presence of Pi. It has been found previously that binding of the epsilon subunit to ECF1 inhibits ATPase activity by decreasing the off rate of Pi [Dunn, S. D., Zadorozny, V. D., Tozer, R. G., & Orr, L. E. (1987) Biochemistry 26, 4488-4493]. This reciprocal relationship between Pi binding and epsilon-subunit conformation has important implications for energy transduction by the E. coli ATP synthase.
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PMID:Catalytic site nucleotide and inorganic phosphate dependence of the conformation of the epsilon subunit in Escherichia coli adenosinetriphosphatase. 182 19

The mechanism by which fluoride and aluminum or beryllium in combination with ADP inhibit beef heart mitochondrial F1-ATPase was investigated. The kinetics of inhibition depended on the nature of the anion present in the F1-ATPase assay medium. Inhibition required the presence of Mg2+ and developed more rapidly with sulfite and sulfate than with chloride, i.e., with anions which activate F1-ATPase activity. The ADP-fluorometal complexes were bound quasi-irreversibly to F1, and each mole of the inhibitory nucleotide-fluorometal complex was tightly associated with 1 mol of Mg2+. One mole of nucleotide-fluorometal complex was able to inhibit the activity of 1 mol of catalytic site in F1. Direct measurements of bound fluoride, aluminum, beryllium, and ADP indicated that the F1-bound ADP-fluorometal complexes are of the following types: ADP1A11F4, ADP1Be1F1, ADP1Be1F2, or ADP1Be1F3. Fluoroaluminates or fluoroberyllates are isomorphous to Pi, and the inhibitory nucleotide-fluorometal complexes mimicked transient intermediates of nucleotides that appeared in the course of ATP hydrolysis. On the other hand, each mole of fully inhibited F1, retained 2 mol of inhibitory complexes. The same stoichiometry was observed when ADP was replaced by GDP, a nucleotide which, unlike ADP, binds only to the catalytic sites of F1. These results are discussed in terms of a stochastic model in which the three cooperative catalytic sites of F1 function in interactive pairs.
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PMID:Fluoroaluminum and fluoroberyllium nucleoside diphosphate complexes as probes of the enzymatic mechanism of the mitochondrial F1-ATPase. 182 93

Bovine heart submitochondrial particles incubated with a low concentration of ADP in the presence of Mg2+ and passed through a Sephadex column equilibrated with EDTA exhibit sensitivity of their initial ATPase activity to preincubation with Mg2+. By using particles thus prepared, several characteristics of a Mg(2+)-specific inhibitory site on F0.F1 ATPase were studied. The inhibition was shown to be both time- and Mg(2+)-concentration-dependent, with an equilibrium constant (at infinite time) of 2 x 10(-6) M (25 degrees C, pH 7.5). The dependence of the pseudo-first-order rate constant for the inhibition process on Mg2+ concentration suggests the presence of a single Mg(2+)-binding site with K8 = 1.1 x 10(-4) M. The data obtained are consistent with a two-step mechanism of Mg(2+)-F0.F1 interaction which results in a loss of the ATPase activity; it includes rapid pH-dependent binding of Mg2+ at the site with K8 = 1.1 x 10(-4) M, followed by a slow interconversion of the Mg(2+)-F1 complex into inactive ATPase (kin. = 0.65 min-1, kact. = 0.01 min-1). The Mg(2+)-inhibited ATPase is very slowly (t1/2 approximately 90 min) re-activated in the presence of EDTA. The rate of EDTA-induced re-activation is pH-independent and can be dramatically increased by added ATP, Pi and sulphite. The dissociation constants for free ATP and P1 (5 x 10(-7) M and 1 x 10(-3) M respectively) and the maximal activation rates were determined by measuring the hyperbolic dependencies of the EDTA-induced re-activation of Mg(2+)-de-activated ATPase on the concentrations of the accelerating ligands. Taken together, the data obtained show two functionally detectable free nucleotide-specific binding sites, one site for Pi and one Mg(2+)-specific ATPase-inhibitory site on the F0.F1 mitochondrial ATP synthase complex.
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PMID:Interaction of Mg2+ with F0.F1 mitochondrial ATPase as related to its slow active/inactive transition. 182 47

The alpha 3 beta 3 complex of ATP synthase obtained from a thermophilic bacterium PS3 was isolated and found to show the ATPase activity (Kagawa, Y., Ohta, S., and Otawara-Hamamoto, Y. (1989) FEBS Lett. 249, 67-69). The structure and the nucleotide binding effects of the alpha 3 beta 3 complex were investigated by means of small-angle x-ray scattering and high performance liquid chromatography. The scattering profile from the alpha 3 beta 3 complex was explained with a model in which the complex is made of an ellipsoid of revolution with the axes of 121.8, 121.8, and 72.0 A having an elliptical hollow cavity with the axes of 35.4, 35.4, and 72.0 A. By the addition of Mg.AT(D)P, significant changes in the scattering profile were observed, in which the radius of gyration decreased from 44 to 35 A. This change was found by gel filtration to be caused by the dissociation reaction from the alpha 3 beta 3 hexamer to the alpha beta dimer. The dissociation of the alpha 3 beta 3 complex was not induced by unhydrolyzable ATP analogue, nor by Pi, Mg2+, and Pi + Mg2+. The structure of the dimer was well explained by the triaxial ellipsoidal model with the axes of 105.2, 39.4, and 108.2 A. The dissociation into the dimer is considered to be related to the ATPase activity because the AT(D)P-induced dissociation is observed only in the presence of Mg2+ ions.
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PMID:Small-angle X-ray scattering studies of Mg.AT(D)P-induced hexamer to dimer dissociation in the reconstituted alpha 3 beta 3 complex of ATP synthase from thermophilic bacterium PS3. 182

The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.
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PMID:The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F1-ATPase. 182 2

Treatment of isolated, latent chloroplast ATPase with pyridoxal-5-phosphate (pyridoxal-P) in presence of Mg2+ causes inhibition of dithiothreitol-activated plus heat-activated ATP hydrolysis. The amount of [3H]pyridoxal-P bound to chloroplast coupling factor 1 (CF1) was estimated to run up to 6 +/- 1 pyridoxal-P/enzyme, almost equally distributed between the alpha- and beta-subunits. Inactivation, however, is complete after binding of 1.5-2 pyridoxal-P/CF1, suggesting that two covalently modified lysines prevent the activation of the enzyme. ADP as well as ATP in presence of Mg2+ protects the enzyme against inactivation and concomittantly prevents incorporation of a part of the 3H-labeled pyridoxal-P into beta- and alpha-subunits. Phosphate prevents labeling of the alpha-subunit, but has only a minor effect on protection against inactivation. The data indicate a binding site at the interface between the alpha- and beta-subunits. Cleavage of the pyridoxal-P-labeled subunits with cyanogen bromide followed by sequence analysis of the labeled peptides led to the detection of Lys beta 359, Lys alpha 176 and Lys alpha 266, which are closely related to proposed nucleotide-binding regions of the alpha- and beta-subunits.
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PMID:Inactivation of chloroplast H(+)-ATPase by modification of Lys beta 359, Lys alpha 176 and Lys alpha 266. 183 78

High concentrations of either Mg-ATP complex, free ATP, or free Mg2+ ions were inhibitors of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus. Free Mg2+ acts as a linear competitive inhibitor with regard to Mg-ATP hydrolysis with a Ki value of 2.8 mM. The inhibition by free ATP was markedly biphasic and thus simple competitive inhibition alone is not sufficient to explain the inhibitory effect. From these results conclusions were drawn about the binding of the substrate, Mg-ATP complex, to the enzyme.
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PMID:Inhibition by excess of free ATP, and free Mg2+ ions of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus. 183 62

The F1 complex of the ATP synthase of Streptomyces lividans was isolated and purified. The procedure involved the solubilization of F1 from membranes with buffer of low ionic strength in the presence of EDTA, ion-exchange chromatography and gel filtration. The purified F1 complex from S. lividans (SLF1) consists of five subunits alpha, beta, gamma, delta and epsilon with molecular masses of 58,000, 50,000, 36,000, 28,000 and 13,000, respectively and exhibits immunological cross-reactivity with the F1 portion purified from Escherichia coli (ECF1). The enzymatic properties of SLF1 were determined by the use of microtiter-plate-based assay and compared with data obtained for ECF1. ATPase activity of SLF1 (specific activity: 20-30 U/mg) was only observed in the presence of high concentrations of Ca2+ (10mM). Stimulation of the ATPase activity by Mg2+ was not detectable; quite to the contrary, Mg2+ inhibited the Ca(2+)-stimulated activity of SLF1. SLF1 was re-bound to F1-stripped membranes of S. lividans, but not to F1-stripped membrane vesicles of E. coli. In contrast, ECF1 could be cross-reconstituted with F1-stripped membranes of S. lividans; however, a structural but not a functional reconstitution of the hybrid F1Fo complex was observed.
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PMID:Purification and characterization of the F1 portion of the ATP synthase (F1Fo) of Streptomyces lividans. 183 70

Mitochondrial F1-ATPase was purified from the mycelium of Phycomyces blakesleeanus NRRL 1555(-) and its kinetic characteristics were studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme reveals five bands (alpha, beta, gamma, delta, and epsilon) characteristic of the F1 portion with apparent molecular weights of 60,000, 53,000, 31,000, 25,000, and 21,000, respectively. The molecular weight of the native F1-ATPase from Phycomyces blakesleeanus was in agreement with the stoichiometry alpha 3 beta 3 gamma delta epsilon. The MgATP complex is the true substrate for ATPase activity which has a Km value of 0.15 mM. High concentrations of free ATP or free Mg2+ ions inhibit the ATPase activity. ADP appears to act as a negative allosteric effector with regard to MgATP hydrolysis, with the apparent Vmax remaining unchanged.
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PMID:Mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus: purification, characterization, and kinetic studies. 183 28

In order to understand the role of carnitine metabolites in the genesis of cellular dysfunction and damage due to myocardial ischemia, the effects of 1-100 microM L-carnitine, acetylcarnitine, propionylcarnitine, and palmitoylcarnitine were investigated on rat heart sarcolemmal, sarcoplasmic reticular, and mitochondrial ATPase activities. Palmitoylcarnitine, unlike acetylcarnitine, propionylcarnitine and carnitine, produced marked inhibitory actions on sarcolemmal Na,K-ATPase and Ca2(+)-stimulated ATPase, as well as sarcoplasmic reticular Ca2(+)-stimulated ATPase activities; Na,K-ATPase was most sensitive. Although palmitoylcarnitine, unlike carnitine or its short-chain fatty-acid derivatives, also depressed sarcolemmal Ca2+ ATPase or Mg2+ ATPase, sarcoplasmic reticular Mg2+ ATPase, and mitochondrial Mg2+ ATPase, mitochondria were less sensitive in comparison to other organelles. Myofibrillar Ca2(+)-stimulated ATPase was slightly inhibited by very high concentrations of palmitoylcarnitine only. It is suggested that the observed depression of the sarcolemmal Na(+)-pump system by low concentrations of long-chain acyl derivatives of carnitine may contribute towards the pathogenesis of arrhythmias due to myocardial ischemia. Furthermore, the inhibition of Ca2(+)-pump mechanisms in the sarcolemmal and sarcoplasmic reticular membranes by relatively high concentrations of palmitoylcarnitine may result in the occurrence of intracellular Ca2+ overload and subsequent cell damage, as well as cardiac dysfunction due to myocardial ischemia.
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PMID:Effects of some L-carnitine derivatives on heart membrane ATPases. 185 32


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