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)

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification of the subunits of F1F0-ATPase from bovine heart mitochondria. 182 92

A novel, simple, and rapid preparative method for purification of rat liver H(+)-ATP synthase by anion-exchange HPLC was developed. The H(+)-ATP synthase purified had higher ATPase activity in the absence of added phospholipids than any preparation reported previously, and this activity was completely inhibited by oligomycin. When reconstituted into proteoliposomes, the H(+)-ATP synthase showed an ATP-dependent 8-anilinonaphthalene-1-sulfonate response and ATP-Pi exchange activity, both of which were also completely inhibited by oligomycin and an uncoupler, indicating the intactness of the H(+)-ATP synthase. An immunochemical study and a labeling experiment with N,N'-[14C]dicyclohexylcarbodiimide ([14C]DCCD) demonstrated the presence of chargerin II ( a product of mitochondrial A6L DNA) and DCCD-binding protein (subunit c) in the complex. The subunits of the complex were separated into 11 main fractions by reverse-phase HPLC, and 3 of them and the delta subunit in F1 were partially sequenced. A search for sequence homologies indicated that these components were subunit b, coupling factor 6, subunit delta, and subunit epsilon. This is the first report of the existence of subunit b, factor 6, and chargerin II in H(+)-ATP synthase purified from rat liver mitochondria.
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PMID:H(+)-ATP synthase from rat liver mitochondria. A simple, rapid purification method of the functional complex and its characterization. 182 63

Dicyclohexylcarbodiimide (DCCD) inhibits the activity of the F1F0-H+ ATP synthase of Escherichia coli by reacting with aspartyl 61 in subunit c of the FO sector to form a stable N-acylurea. The segment of chromosomal DNA which codes the subunits of the FO was cloned from four independently isolated DCCD-resistant mutants, and the sequence of the subunit c gene (uncE) was determined. An Ala24 to serine (A24S) substitution was found in the subunit c gene of each mutant. The A24S uncE gene was cloned into the BamHI site of a mutant derivative of plasmid pBR322. The A24S subunit c conferred DCCD resistance to a variety of recipient E. coli strains when it was overexpressed from this plasmid. A 7-base pair deletion beginning at position 132 of the plasmid vector was responsible for the observed overexpression. Hoppe et al. (Hoppe, J., Schairer, H. U., and Sebald, W. (1980) Eur. J. Biochem. 112, 17-24) had previously shown that mutation of subunit c Ile28 to threonine or valine resulted in DCCD resistance. The DCCD sensitivities of the membrane ATPase of these mutants and the A24S mutant were compared. DCCD sensitivity decreased in the order: wild-type much greater than I27V greater than I28T = A24S. The venturicidin sensitivities of wild-type and mutant membranes were also examined. The membrane ATPase of the I28T and I28V mutants was venturicidin resistant whereas the A24S substitution resulted in a hypersensitivity to inhibition by venturicidin. These results support a model in which subunit c folds in the membrane like a hairpin, where the region of residues 24-28 in transmembrane helix-1 is close to that of aspartyl 61 in transmembrane helix-2.
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PMID:Mutation of alanine 24 to serine in subunit c of the Escherichia coli F1F0-ATP synthase reduces reactivity of aspartyl 61 with dicyclohexylcarbodiimide. 183 53

1. Peroxisomes were isolated from bovine and rat liver by use of differential and density gradient centrifugations. 2. In the final density gradient (Nycodenz) a distinct peak of ATPase activity codistributed with the peroxisome marker catalase and was well separated from the bulk of the ATPase activity and from markers for other subcellular organelles. 3. The peroxisome-associated ATPase had a pH optimum of 7.5 and was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was unaffected by up to 30 microM n-tributyltin chloride. 4. Prolonged incubation with oligomycin at high concentrations indicated that 50% of peroxisomal ATPase was resistant to this inhibitor. The oligomycin-sensitive ATPase activity required at least a four-fold higher ratio of inhibitor to protein for inhibition than mitochondrial ATPase did. It was concluded that oligomycin-sensitive and oligomycin-resistant ATPase may be associated with liver peroxisomes.
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PMID:Properties of ATPase activity associated with peroxisomes of rat and bovine liver. 183 59

The involvement of ATP synthase in the imbalance between the photoactivities of PS I and PS II under light-limiting conditions, was examined in broken lettuce chloroplasts using modulated fluorimetry. The imbalance, in favor of PS II, was minimal and roughly constant between pH 6.5-7.3 (ratio of PS II/PS I activities about 1.1), and maximal at pH 8.5 (ratio of PS II/PS I activities about 1.4). This increase was strongly inhibited by a treatment of the chloroplasts with the CF0 ATP synthase inhibitor DCCD, but unaffected by the CF1 ATPase inhibitor, tentoxin. However, tentoxin plus ADP-P1 did inhibit the high pH-induced increased imbalance. These results, when considered with the previous results on the effect of high pH on proton flux through the ATP synthase, suggest that the rate of such proton flow controls the imbalance between the two photo-systems. It is possible that there is an in vivo fine-tuning regulating mechanism of the photosystems imbalance via the opening and closing of proton gradient dissipation through the ATP synthase. This mechanism may help alleviate photoinhibitory damage.
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PMID:Proton flow through the ATP synthase in chloroplasts regulates the distribution of light energy between PS I and PS II. 184 95

The three beta subunits of the isolated Escherichia coli F1-ATPase react independently with chemical reagents (Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 284, 116-120). Thus, one beta subunit is readily cross-linked to the epsilon subunit, Another reacts with N,N'-dicyclohexylcarbodiimide (DCCD), and the third one is modified on a lysine residue by 4-chloro-7-nitrobenzofurazan (NbfCl). The binding site for the ATP analog, 2-azido-ATP, was not associated with a specific type of beta subunit (Bragg, P.D. and Hou, C. (1989) Biochim. Biophys. Acta 974, 24-29). We now show that this binding site is a catalytic site as opposed to a noncatalytic nucleotide-binding site. NbfCl reacted with a tyrosine residue on the DCCD-reacting beta subunit in contrast to the different subunit location of the lysine residue labeled by the reagent. Thus, O to N transfer of the Nbf group in the free F1-ATPase involves transfer between subunits. The chemical labelling pattern of membrane-bound F1-ATPase differed from that of free F1. The strict asymmetry of labeling of the free F1-ATPase was not observed. Thus, double labeling of beta subunits by several reagents was found. This suggests that the asymmetry was not induced by chemical modification, but is inherent in the structure of the ATPase.
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PMID:Reaction of membrane-bound F1-adenosine triphosphatase of Escherichia coli with chemical ligands and the asymmetry of beta subunits. 213 13

An ATPase from anaerobic Lactobacillus casei has been isolated and 100-times purified. The 400 kDa enzyme molecule was found to have a hexagonal structure 10 nm in diameter composed of at least six protein masses. SDS-electrophoresis reveals four or, under certain conditions, five types of subunit, of apparent molecular masses 57 (alpha), 55 (beta), 40 (gamma), 22 (delta) and 14 (epsilon) kDa with stoichiometry of 3 alpha, 3 beta, gamma, delta, epsilon. The following features resembling F1-ATPases from other sources were found to be inherent in the solubilized L. casei ATPase. (i) Detachment from the membrane desensitizes ATPase to low DCCD concentrations and sensitizes it to water-soluble carbodiimide. (ii) Soluble ATPase is inhibited by Nbf chloride and azide, is resistant to SH-modifiers and is activated by sulfite and octyl glucoside, the activating effect being much stronger than in the case of the membrane-bound ATPase. Substrate specificity of the enzyme is also similar to that of other factors F1. Divalent cations strongly activate the soluble enzyme when added at a concentration equal to that of ATP. An excess of Mn2+, Mg2+ or Co2+ inhibits ATPase activity of F1, whereas that of Ca2+ induces its further activation. No other F1-like ATPases are found in L. casei. It is concluded that this anaerobic bacterium possesses a typical F1-ATPase similar to those in mitochondria, chloroplasts, aerobic and photosynthetic eubacteria.
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PMID:The F1-type ATPase in anaerobic Lactobacillus casei. 213 82

Chloroplast F0 (CF0) was purified from the ATP synthase by Zwittergent 3-12 treatment and DEAE-Trisacryl anion exchange chromatography. Purified CF0 contains four subunits corresponding to subunits I, II, III, and IV. CF0 mediated proton translocation across the membrane after incorporation into asolectin liposomes. The CF0-mediated proton transport was inhibited by N,N'-dicyclohexylcarbodiimide and the binding of chloroplast coupling factor 1 (CF1). Rebinding of CF1 to CF0 liposomes resulted in reconstitution of N,N'-dicyclohexylcarbodiimide and uncoupler sensitive energy-transducing activities. Like CF0 in native thylakoid membranes, purified CF0 bound CF1 as well as CF1 deficient in either the delta or epsilon subunits.
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PMID:Purification and reconstitution of active chloroplast F0. 215 62

The F1F0-ATP synthase from the alkaliphilic Bacillus firmus OF4 was purified in a reconstitutively active form, in good yield and with a high specific ATPase activity when appropriately activated. The purification procedure involved octyl glucoside extraction of washed membrane vesicles in the presence of 20% glycerol and asolectin followed by ammonium sulfate fractionation and sucrose density gradient centrifugation. The purified preparation was resolved into seven bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to the five F1 subunits, alpha, beta, gamma, delta, and epsilon, and to the b and c subunits of the F0. Two-dimensional sodium dodecyl sulfate-poly-acrylamide gel analysis revealed a candidate for the alpha subunit of F0. The MgATPase activity of B. firmus OF4 F1F0 was barely detectable but could be stimulated, optimally more than 100-fold, by sulfite, methanol, and octyl thioglucoside. The enzyme was inhibited by N,N'-dicyclohexylcarbodiimide and sodium azide, but not by aurovertin, an inhibitor of the F1 from Escherichia coli. The F1F0 reconstituted into proteoliposomes catalyzed ATPase activity, ATP-Pi exchange, and ATP-dependent delta pH and delta psi formation. ATP hydrolysis was stimulated by protonophores while the other activities were abolished by protonophores. These activities were neither dependent on added sodium ions nor significantly affected by them. F1F0 proteoliposomes made from crude octyl glucoside extracts that also contained the Na+/H+ antiporter were shown to catalyze ATP-dependent Na+ uptake that was completely sensitive to carbonyl cyanide m-chlorophenyl-hydrazone; Na+ uptake activity was absent in proteoliposomes containing more purified F1F0 but lacking the Na+/H+ antiporter. These data show that the F1F0 translocates protons and does not substitute Na+ for H+ in energy coupling.
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PMID:Purification and reconstitution of the F1F0-ATP synthase from alkaliphilic Bacillus firmus OF4. Evidence that the enzyme translocates H+ but not Na+. 217 11

The energy-transducing mechanism of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius DSM 639 has been studied, addressing the question whether chemiosmotic proton gradients serve as an intermediate energy store driving an F0F1-analogous ATP synthase. At pH 3.5, respiring S. acidocaldarius cells developed an electrochemical potential of H+ ions, consisting mainly of a proton gradient and a small inside-negative membrane potential. The steady-state proton motive force of 140 to 160 mV was collapsed by protonophores, while N,N'-dicyclohexylcarbodiimide (DCCD) caused a hyperpolarization of the membrane, as expected for a reagent commonly used to inhibit the flux through proton channels of F0F1-type ATP synthases. Cellular ATP content was strongly related to the proton motive force generated by respiration and declined rapidly, either by uncoupling or by action of DCCD, which in turn induced a marked respiratory control effect. This observation strongly supports the operation of chemiosmotic ATP synthesis with H+ as the coupling ion. The inhibition of ATP synthesis by [14C]DCCD was correlated with covalent reactions with membrane proteins. The extraction of labeled membranes with organic solvents specifically yielded a readily aggregating proteolipid of 6 to 7 kilodaltons apparent molecular mass. Its amino acid composition revealed significant similarity to the proteolipid found in eubacteria, such as Escherichia coli, as an extremely hydrophobic constituent of the F0 proton channel. Moreover, the N-terminal amino acid sequence of the Sulfolobus proteolipid displays a high degree of homology to eubacterial sequences, as well as to one derived from nucleic acid sequencing of another Sulfolobus strain (K. Denda, J. Konishi, T. Oshima, T. Date, and M. Yoshida, J. Biol. Chem. 264:7119-7121, 1989). Despite certain structural similarities between eucaryotic vacuolar ATPases and the F1-analogous ATPase from Sulfolobus sp. described earlier, the results reported here promote the view that the archaebacterial ATP-synthesizing complex functionally belongs to the F0F1 class of ATPases. These may be considered as phylogenetically conserved catalysts of energy transduction present in all kingdoms of organisms.
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PMID:Chemiosmotic energy conversion of the archaebacterial thermoacidophile Sulfolobus acidocaldarius: oxidative phosphorylation and the presence of an F0-related N,N'-dicyclohexylcarbodiimide-binding proteolipid. 247 23


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