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 interaction of inorganic phosphate with native and nucleotide-depleted F1-ATPase was studied. F1-ATPase depleted of tightly bound nucleotides loses the ability to bind inorganic phosphate. The addition of ATP, ADP, GTP and GDP but not AMP, restores the phosphate binding. The nucleotides affecting the phosphate binding to F1-ATPase are located at the catalytic (exchangeable) site of the enzyme. The phosphate is thought to bind to the same catalytic site where the nucleotide is already bound. It is thought that ADP is the first substrate to bind to F1-ATPase in the ATP synthesis reaction.
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PMID:Tightly bound nucleotides affect phosphate binding to mitochondrial F1-ATPase. 285 8

Bidentate cobalt(III)tetraamine adenosine triphosphate [Co(NH3)4ATP] was investigated as an inhibitor of the beef heart mitochondrial F1-ATPase. The compound was found to have a mixed noncompetitive mechanism with a Ki of 0.4 mM and an alpha of 1.4 during ATP hydrolysis. Co(NH3)4ATP also noncompetitively inhibited ATP hydrolysis in the presence of bicarbonate. ITP hydrolysis was similarly affected. Co(NH3)4ATP was also used in dual inhibitor studies with adenylylimidodiphosphate (AMP-PNP) and azide; it was found to be mutually exclusive with AMP-PNP and azide. The compound also protected the F1 from modification by 4-chloro-7-nitrobenzofurazan. These results are discussed in terms of the regulation of the ATP hydrolysis reaction.
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PMID:The effect of Co(III)(NH3)4ATP on the kinetics of beef heart mitochondrial ATPase. 285 40

The possibility that 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive derivative of inorganic phosphate (Pi) [Lauquin, G., Pougeois, R., & Vignais, P. V. (1980) Biochemistry 19, 4620-4626], could mimic ATP was investigated. ANPP was hydrolyzed in the dark by sarcoplasmic reticulum Ca2+-ATPase in the presence of Ca2+ but not in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANPP was not hydrolyzed by purified mitochondrial F1-ATPase; however, ADP and ATP protected F1-ATPase against ANPP photoinactivation. On the other hand, the trinitrophenyl nucleotide analogues (TNP-ADP, TNP-ATP, and TNP-AMP-PNP), which bind specifically at the two catalytic sites of F1-ATPase [Grubmeyer, C., & Penefsky, H. (1981) J. Biol. Chem. 256, 3718-3727], abolished Pi binding on F1-ATPase; they do not protect F1-ATPase against ANPP photoinactivation. Furthermore, ANPP-photoinactivated F1-ATPase binds the TNP analogues in the same way as the native enzyme. The Pi binding site of F1-ATPase, which is shown to be photolabeled by ANPP, does not appear to be at the gamma-phosphate position of the catalytic sites.
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PMID:Further investigations on the inorganic phosphate binding site of beef heart mitochondrial F1-ATPase. 285 84

The native tonoplast and the mitochondrial H+-ATPase from oat roots were compared to determine whether the two enzymes have similar mechanisms. H+ pumping in low-density microsomal vesicles reflected activity from the tonoplast-type ATPase, as ATPase activity and ATP-dependent H+ pumping (quinacrine fluorescence quenching) showed similar sensitivities to inhibition by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonate, nitrate, quercetin, or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. The tonoplast-type ATPase was stimulated by C1-,Br- greater than HCO3- whereas the mitochondrial ATPase was stimulated by HCO3- much greater than C1-,Br-. Both enzymes hydrolyzed ATP preferentially and were inhibited competitively by AMP or ADP. Apart from resistance to azide, the tonoplast-type ATPase was strikingly similar in its inhibitor sensitivities to the mitochondrial ATPase. The insensitivity to vanadate of both enzymes suggests the reaction mechanisms do not involve a covalent phosphoenzyme. Inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide and protection by ATP suggests tyrosine and cysteine residues are in the catalytic site of the tonoplast ATPase. The mitochondrial ATPase was 100 times more sensitive to N,N'-dicyclohexyl-carbodiimide inhibition than the tonoplast H+-ATPase. These results suggest the tonoplast and the mitochondrial H+-ATPases share common steps in their catalytic and vectorial reaction mechanisms, yet sufficient differences exist to indicate they are two distinct ATPases.
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PMID:Similarities and differences between the tonoplast-type and the mitochondrial H+-ATPases of oat roots. 286 67

Beef heart mitochondrial ATPase (F1) catalyzes the hydrolysis of the ATP analog adenyl-5-yl imidodiphosphate (AMP-PNP). The reaction products are inorganic phosphate and adenyl-5-yl phosphoramidate (AMP-PN) as determined by HPLC analysis. The hydrolysis occurs in both the presence and absence of added divalent metal ions and is stimulated by potassium. The kinetic properties of the hydrolytic reaction depend markedly on the identity of the added divalent metal. GMP-PNP and AMP-CPP are also hydrolyzed, while AMP-PCP is not. Adenyl-5-yl phosphoramidate is a potent effect of beef heart mitochondrial ATPase activity. Based on these data, a reinterpretation of work based on the assumption that AMP-PNP is not hydrolyzed is presented.
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PMID:Hydrolysis of adenyl-5-yl imidodiphosphate by beef heart mitochondrial ATPase. 286 12

F1-ATPase of rat liver was examined for its capacity to interact with both metal ions and nucleotides and for the effect of covalent ATPase inhibitors on these interactions. As isolated, rat liver F1 contains about 2 mol of Mg2+/mol of F1, 1 mol of which can be removed or exchanged. The remaining mole of Mg2+ per mole of F1 remains very tightly associated with F1 and is recovered in the alpha gamma fraction after cold denaturation. Rat liver F1 also contains as isolated a nearly equivalent amount of nucleotide (approximately 1.7 mol/mol of F1) which is readily removed by incubation at room temperature followed by column centrifugation. The "2 Mg2+ enzyme" binds almost 3 mol of 5'-adenylyl imidodiphosphate (AMP-PNP)/mol of F1 in the presence or absence of added divalent cation. When divalent cation is present as Co2+, an equivalent activator to Mg2+ in the ATPase reaction, 1 mol of F1 binds 3 mol of both AMP-PNP and Co2+. under these conditions, the very tight Mg2+ site remains loaded, the exchangeable Mg2+ site is replaced with AMP-PNPCo, and two additional AMP-PNPCo sites are filled. At this point, ADP can be loaded onto the enzyme as a fourth nucleotide at a site separate and distinct from the AMP-PNP sites. Significantly, rat liver F1 contains only a single readily detectable ADP binding site in the presence or absence of divalent cation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ligand binding studies of the F1 moiety of rat liver ATP synthase: implications about the enzyme's structure and mechanism. 288 76

The Mg2+-ATPase activities of bovine adrenal chromaffin granules were studied in highly purified preparations of granule ghosts and in intact organelles. The overall ATPase activity (150-250 nmol ADP min-1 mg-1) of the granule ghost preparations was inhibited less than 5% by the bathophenanthroline chelate of Fe(II), a potent inhibitor of mitochondrial F1-ATPase. This small inhibition can be accounted for by a very minor contamination with mitochondria or mitochondrial fragments. The overall ATPase activity of native granule ghosts was inhibited about 75% by N-ethylmaleimide, with half-maximal inhibition at about 20 microM. The titration curve was slightly shifted towards higher concentrations as compared to the inhibition curve for the proton pump activity, which was completely inhibited at 25 microM. N,N'-Dicyclohexylcarbodiimide inhibited the overall ATPase activity by 75-80% at 1.1 mumol/mg protein, a concentration that completely abolished the proton pump activity. Low concentrations (10 microM) of vanadate inhibited the overall ATPase activity by about 15% but had no effect on the proton pump activity, which was partly inhibited only at higher vanadate concentrations. Our attempts to assign a function to the vanadate-sensitive and N-ethylmaleimide-insensitive ATPase have so far been unsuccessful. In particular, our assay for ATP diphosphohydrolase activity was negative, although the chromaffin granule ghosts revealed a low Mg2+-ADPase activity (11.8 nmol AMP min-1 mg-1 protein). In intact chromaffin granules the specific Mg2+-ATPase activity (50-70 nmol ADP min-1 mg-1) was stimulated 2-fold by uncouplers, as compared to 1.6-1.7-fold in granule ghosts. The degree of energy coupling was rather independent of the external pH (6.5 less than pH less than 8.0) and temperature (20-45 degrees C). As expected, partial inhibition (about 15%) of the overall ATPase activity by 10 microM vanadate increased the ATPase control ratio. ADP was found to be a potent inhibitor of the proton pump activity with MgATP as the substrate, and the effect can partly be explained by a competitive type of inhibition of the hydrolytic reaction. This effect of ADP explains some of the kinetic data reported for MgATP-dependent (H+-ATPase-dependent) reactions in this organelle, notably the energy-dependent accumulation and storage of catecholamines.
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PMID:Studies on Mg2+-dependent ATPase in bovine adrenal chromaffin granules. With special reference to the effect of inhibitors and energy coupling. 288 84

The effect of exposure to adenine nucleotides on the dissociation of phosphate bound to beef heart mitochondrial F1-ATPase (F1) has been studied using a layered Sephadex centrifuge column method. The order of effectiveness of nucleotides in facilitating Pi dissociation from F1 was ATP greater than AMP-PNP much greater than ADP (where AMP-PNP indicates adenosine 5'-(beta,gamma-imido)triphosphate) when phosphate had bound to F1 as Pi from the medium. When ATP was present in amounts substoichiometric to F1, essentially all of the ATP present bound to F1, and the molar ratio of Pi released to ATP bound was near one. Under similar conditions AMP-PNP bound equally well to F1, but in order to effect similar amounts of Pi release it was necessary to use approximately 10 times as much AMP-PNP as ATP. The order of effectiveness of nucleotides in facilitating dissociation of phosphate which had bound to F1 as the gamma-phosphate of ATP was ATP = AMP-PNP much greater than ADP. In this case ATP and AMP-PNP were as effective in facilitating phosphate dissociation as was ATP in the case in which phosphate had bound to F1 as Pi. It is concluded that when phosphate has bound to F1 as Pi, binding of one molecule of ATP or more than one molecule of AMP-PNP are sufficient to facilitate dissociation of phosphate. When phosphate has bound to F1 as the gamma-phosphate of ATP, binding of one molecule of either ATP or AMP-PNP is sufficient to facilitate dissociation of phosphate.
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PMID:Dissociation of phosphate from beef heart mitochondrial F1-ATPase. Effect of adenine nucleotides. 288

The stoichiometry of nucleotide binding to the isolated alpha- and beta-subunits of Escherichia coli F1-ATPase was investigated using two experimental techniques: (a) titration with fluorescent trinitrophenyl (TNP) derivatives of AMP, ADP, and ATP and (b) the centrifuge column procedure using the particular conditions of Khananshvili and Gromet-Elhanan (Khananshvili, D., and Gromet-Elhanan, Z. (1985) FEBS Lett. 178, 10-14). Both procedures showed that alpha-subunit contains one nucleotide-binding site, confirming previous work. TNP-ADP and TNP-ATP bound to a maximal level of 1 mol/mol beta-subunit, consistent with previous equilibrium dialysis studies which showed isolated beta-subunit bound 1 mol of ADP or ATP per mol (Issartel, J. P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). However, binding of only approximately 0.1 mol of ATP or ADP per mol of beta-subunit was detected using centrifuge columns. Our results are consistent with the conclusion that each of the alpha- and beta-subunits contains one nucleotide-binding domain. Because the subunit stoichiometry is alpha 3 beta 3 gamma delta epsilon, this can account for the location of the six known nucleotide-binding sites in E. coli F1-ATPase. Studies of in vitro assembly of isolated alpha-, beta-, and gamma- subunits into an active ATPase showed that ATP, GTP, and ITP all supported assembly, with half-maximal reconstitution of ATPase occurring at concentrations of 100-200 microM, whereas ADP, GDP, and IDP did not. Also TNP-ATP supported assembly and TNP-ADP did not. The results demonstrate that (a) the nucleotide-binding site on beta-subunit has to be filled for enzyme assembly to proceed, whereas occupancy of the alpha-subunit nucleotide-binding site is not required, and (b) that enzyme assembly requires nucleoside triphosphate.
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PMID:Trinitrophenyl-ATP and -ADP bind to a single nucleotide site on isolated beta-subunit of Escherichia coli F1-ATPase. In vitro assembly of F1-subunits requires occupancy of the nucleotide-binding site on beta-subunit by nucleoside triphosphate. 289 69

Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), an adenine nucleotide affinity analog, was found to bind in a saturable fashion to isolated alpha-subunit from Escherichia coli F1-ATPase with a stoichiometry of one mol/mol and a Kd approximately 150 microM. The binding was shown to be specific by the following criteria: 1) ATP reduced the binding of PLP-AMP by 80%, and 2) PLP-AMP, like ATP, induced a conformational change which increased the mobility of alpha-subunit in nondenaturing polyacrylamide gel electrophoresis and rendered alpha-subunit resistant to mild trypsin proteolysis. A stable adduct was formed between isolated alpha-subunit and [3H] PLP-AMP after reduction with NaBH4. alpha-Subunit labeled to the extent of 0.4-0.7 mol/mol was digested with trypsin and subjected to high pressure liquid chromatography purification, yielding a single labeled peptide. Automated amino acid sequencing showed that residue alpha-Lys-201 was specifically labeled. The results suggest that Lys-201 occupies a position proximate to the phosphate groups of bound ATP in the alpha.ATP complex. PLP-AMP did not support repolymerization of isolated alpha-, beta-, and gamma-subunits, consistent with previous reports that subunit repolymerization in vitro is dependent upon the presence of nucleoside triphosphate. Further, PLP-AMP-labeled alpha-subunit could not be reconstituted with isolated beta- and gamma-subunits in the presence of ATP, showing that occupation of the alpha-subunit nucleotide site by PLP-AMP impairs normal subunit-subunit interaction.
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PMID:Pyridoxal 5'-diphospho-5'-adenosine binds at a single site on isolated alpha-subunit from Escherichia coli F1-ATPase and specifically reacts with lysine 201. 289 72


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