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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)

ADP-induced inhibition of mitochondrial F1-ATPase has been studied. It is shown that in the presence of magnesium and the absence of light, the photoaffinity ADP analog, 2-azido-ADP, induces a reversible inhibition of native F1 that is indistinguishable from that obtained with ADP. Photolysis of the inactive complex results in the predominant labeling of a catalytic-site peptide identified previously (Cross et al., 1987, Proc. Natl. Acad. Sci. USA 84, 5715-5719). Dissociation of the inactive complex formed between F1 and ADP is biphasic with a rapid azide-insensitive phase followed by a slow azide-sensitive phase (k approximately 3 x 10(-3) s-1). It is also shown that incubation of the ADP-inhibited enzyme with EDTA or phosphate does not result in release or migration of ADP from the catalytic site. However, it does convert the complex to a form that reactivates in the presence of 100 microM ATP at a rate too rapid to observe using manual mixing.
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PMID:Adenine nucleotide-binding sites on mitochondrial F1-ATPase: studies of the inactive complex formed upon binding ADP at a catalytic site. 153

(1) Previous mutational analyses have shown that residue beta R398 of the beta-subunit is a key residue for binding of the inhibitory antibiotic aurovertin to Escherichia coli F1Fo-ATP synthase. Here, we studied purified F1 from the beta R398C and beta R398W mutants. ATPase activity in both cases was resistant to aurovertin inhibition. The fluorescence spectrum (lambda exc = 278 or 295 nm) of beta R398W F1 showed a significant red-shift as compared to wild-type and beta R398C enzymes, indicating that residue beta R398 lies in a polar environment. On the basis of this and previous evidence, we propose that aurovertin binding to F1-ATPase involves a specific charged donor-acceptor H-bond between residue beta R398 and the 7-hydroxyl group of aurovertin. (2) The fluorescent substrate analog lin-benzo-ADP was shown to bind to beta R398W F1 catalytic sites with the same Kd values as to wild-type F1, and with the same quenching of the fluorescence of the analog. Fluorescence energy transfer was seen between the beta R398W residue and bound lin-benzo-ADP. Analysis of transfer efficiency at varying stoichiometry of bound lin-benzo-ADP showed that interaction occurred between one beta R398W residue and one catalytic-site-bound analog molecule at a distance of approximately 23 A. The relationships of the aurovertin and catalytic sites in the primary and tertiary structure are discussed.
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PMID:Investigation of the aurovertin binding site of Escherichia coli F1-ATPase by fluorescence spectroscopy and site-directed mutagenesis. 153 96

The dissociation of mitochondrial F1-ATPase with 3 M LiCl at 0 degrees C, followed by reconstitution, has been analysed. FPLC over a gel filtration column in the dissociation buffer revealed the presence of two protein moieties, an alpha 3 gamma delta epsilon complex and single beta-subunits. When the dissociation and chromatography is performed at pH 6.2, the former protein moiety still contains some adenine nucleotides. Reconstitution of the dissociated complex is not possible any more after FPLC, probably due to the loss of residual adenine nucleotides. After a single column centrifugation step one nucleotide per F1 still remains bound. For reconstitution, additional ATP, or a suitable analog, is required. 2-Azido-ATP, but not 8-azido-ATP or ITP, can replace ATP during the reconstitution. F1, reconstituted in the presence of 2-azido-ATP, contains three tightly bound nucleotides, similar to freshly isolated F1, of which in this case one is an adenine nucleotide and two are azido-adenine nucleotides. One of the latter can be rapidly exchanged and is bound to a catalytic site. Covalent binding (at a beta-subunit) of the other tightly bound 2-azido-ATP by ultraviolet illumination does not result in inhibition of the enzyme. Digestion of F1 with trypsin, followed by HPLC, showed that the label is not bound to the fragment containing Tyr-368, nor to the fragment containing Tyr-345. This result was confirmed by CNBr digestion, followed by SDS-urea PAGE. We conclude that during dissociation of F1 one tightly bound nucleotide (ADP) remains bound at an alpha/beta interface site and that for reconstitution binding of ATP to a (non-catalytic) beta-site is required. The conformation of this site differs from that of the two catalytic beta-sites.
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PMID:Characteristics of the non-exchangeable nucleotide binding sites of mitochondrial F1 revealed by dissociation and reconstitution with 2-azido-ATP. 153 23

The binding of ATP radiolabeled in the adenine ring or in the gamma- or alpha-phosphate to F1-ATPase in complex with the endogenous inhibitor protein was measured in bovine heart submitochondrial particles by filtration in Sephadex centrifuge columns or by Millipore filtration techniques. These particles had 0.44 +/- 0.05 nmol of F1 mg-1 as determined by the method of Ferguson et al. [(1976) Biochem. J. 153, 347]. By incubation of the particles with 50 microM ATP, and low magnesium concentrations (less than 0.1 microM MgATP), it was possible to observe that 3.5 mol of [gamma-32P]ATP was tightly bound per mole of F1 before the completion of one catalytic cycle. With [gamma-32P]ITP, only one tight binding site was detected. Half-maximal binding of adenine nucleotides took place with about 10 microM. All the bound radioactive nucleotides were released from the enzyme after a chase with cold ATP or ADP; 1.5 sites exchanged with a rate constant of 2.8 s-1 and 2 with a rate constant of 0.45 s-1. Only one of the tightly bound adenine nucleotides was released by 1 mM ITP; the rate constant was 3.2 s-1. It was also observed that two of the bound [gamma-32P]ATP were slowly hydrolyzed after removal of medium ATP; when the same experiment was repeated with [alpha-32P]ATP, all the label remained bound to F1, suggesting that ADP remained bound after completion of ATP hydrolysis. Particles in which the natural ATPase inhibitor protein had been released bound tightly only one adenine nucleotide per enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding of adenine nucleotides to the F1-inhibitor protein complex of bovine heart submitochondrial particles. 161 Aug 24

The specificity of the mitochondrial and chloroplast processing enzymes for the nuclear-encoded precursor proteins was investigated. Mitochondrial precursor proteins of the Nicotiana plumbaginifolia and the Neurospora crassa beta subunits of F1-ATPase and the Neurospora Rieske FeS precursor protein were processed to the correct mature size by matrix extracts isolated from spinach leaves, yeast, rat liver and beef heart. The mitochondrial extracts failed to process chloroplast precursor proteins of the stromal small subunit of ribulose 1,5-bisphosphate carboxylase and the thylakoid 33 kDa protein of the oxygen-evolving complex. Both mitochondrial F1 beta precursors were specifically processed by a soluble stromal extract from chloroplasts. However, no processing of the Rieske FeS precursor protein was observed under the same conditions with the chloroplast extract. The cleavage of the mitochondrial F1 beta precursors by the chloroplast extract was shown to be sensitive to the metal chelators EDTA and ortho-phenanthroline. The cleavage site of the mitochondrial F1 beta precursor by the chloroplast soluble extract appears to be located at the N-terminus.
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PMID:Specificity of leaf mitochondrial and chloroplast processing systems for nuclear-encoded precursor proteins. 165 54

The effect of the expression of the chloroplast F1-ATPase beta-subunit in two Escherichia coli beta-subunit mutant strains was investigated. The amount of chloroplast beta-subunit formed in E. coli was increased by introducing a 'Shine-Dalgarno' sequence upstream from the translation start site. The chloroplast beta-subunit was membrane bound but was unable to functionally replace the mutant beta-subunit in a strain carrying the uncD409 allele [corrected]. However, in an E. coli mutant strain unable to form the beta- and epsilon-subunits the presence of the chloroplast beta-subunit enabled the assembly of a functional proton pore [corrected]
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PMID:The chloroplast beta-subunit allows assembly of the Escherichia coli F0 portion of the energy transducing adenosine triphosphatase. 165 29

A cDNA library constructed from poly(A)-rich RNA of the sweet potato tuberous root using a newly developed plasmid vector carrying tac-SP6 promoters was used to identify full length cDNAs for the nuclear-encoded delta-subunit of mitochondrial F1-ATPase by oligonucleotide-hybridization selection. Selected clones contained cDNA insert which carry the entire coding capacity for the pre-delta-subunit, since the RNA transcribed in vitro from SP6 promoter on the vector directed the synthesis of pre-delta-subunit polypeptide in a wheat germ in vitro translation assay. The nucleotide sequence of one of these cDNAs indicates that it can code for the pre-delta-subunit of 244 amino acids of which 199 amino acids encode the mature subunit. The amino acid sequence of the mature delta-subunit shows similarities of about 18-25% amino acid positional identity with the delta-subunits of bacterial F1-ATPases, about 26% with the delta-subunit of chloroplast CF1-ATPase, and about 32-37% with oligomycin sensitivity conferring proteins of animal and fungal mitochondria. The N-terminal presequence of the precursor composed of maximum of 45 amino acids does not show any obvious sequence homology with either the transit peptide of the nuclear-encoded pre-delta-subunit of chloroplast CF1 or the presequence of the nuclear-encoded pre-oligomycin sensitivity conferring proteins. At least two types of the delta-subunit cDNAs with very similar structures were identified from the library, and the presence of multiple copies of the delta-subunit gene in the hexaploid genome of the sweet potato is also suggested by genomic Southern blot hybridization.
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PMID:Primary structure of a precursor for the delta-subunit of sweet potato mitochondrial F1-ATPase deduced from full length cDNA. 169 Jul 22

Cytoplasmic male-sterile lines CMS89 and CMSBaso of sunflower (Helianthus annuus) differ from the fertile lines HA89 and Baso in a mitochondrial DNA sequence in the vicinity of the atpA gene. In addition, the transcriptional pattern of the atpA gene is changed in male-sterile lines compared to fertile ones. Besides one main transcript in the fertile lines, the male-sterile lines additionally show larger transcripts. Investigation of Baso and CMSBaso revealed that the two fertility-restored lines of CMS89 have the same transcripts as CMSBaso or a combination of CMSBaso and CMS89. Comparing the mitochondrial in organello translation products we observed a unique 16 kDa protein, which is expressed in male-sterile lines carrying the H. petiolaris cytoplasm but is not detectable in fertile lines with H. annuus cytoplasm. The 16 kDa protein can also be observed in restored lines but not in H. petiolaris. As the expression of the 16 kDa polypeptide seems to be linked to the interspecific cross between H. petiolaris and H. annuus it may play a role in CMS. By different criteria such as molecular mass, isoelectric point and peptide fingerprinting the alpha subunit of the F1-ATPase of male-sterile and fertile lines is very similar if not identical.
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PMID:A mitochondrial 16 kDa protein is associated with cytoplasmic male sterility in sunflower. 171 23

The nuclear gene atp1 encoding the mitochondrial ATP synthase alpha subunit of the fission yeast Schizosaccharomyces pombe was sequenced. It contains a 1,608-base pair-long open reading frame interrupted by two introns of 175 and 269 base pairs, located near the 5'-end of the gene. The initiation site of transcription AAAC was located 60 nucleotides upstream of the translation initiation codon. The deduced polypeptide sequence contains a 27-amino acid residue presequence, presumably involved in mitochondrial targeting, preceding a mature protein of 509 amino acid residues. The atp1 alleles from mutant A2313 (Bouty, M., and Goffeau, A. (1982) Eur. J. Biochem. 125, 471-477) and its related phenotypic revertant R351 (Falson, P., Di Pietro, A., Darbouret, D., Jault, J. M., Gautheron, D. C., Boutry, M., and Goffeau, A. (1987) Biochem. Biophys. Res. Commun. 148, 1182-1188) were also cloned and sequenced. A single nonsense mutation CAA-TAA (Gln173-stop) in mutant A2313 became a missense mutation TAA-TTA (stop-Leucine) in revertant R351. Glutamine 173 is located in the first putative element of the nucleotide binding site. Its substitution by a leucine residue appears responsible for the lower enzyme affinity toward ADP and for the loss of cooperativity of F1-ATPase activity.
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PMID:Alpha subunit of mitochondrial F1-ATPase from the fission yeast. Deduced sequence of the wild type and identification of a mutation that alters apparent negative cooperativity. 182 97

Isolated rat liver mitochondria were incubated in the presence of a reconstituted malate-aspartate shuttle under carboxylating conditions in the presence of glutamate, octanoyl-carnitine and pyruvate, or a preset lactate/pyruvate ratio. The respiration and attendant energy state were varied with soluble F1-ATPase. Under these conditions reducing equivalents are exported due to pyruvate carboxylation. This was shown by lactate production from pyruvate and by a substantial increase in the lactate/pyruvate ratio. This led to a competition between malate export and energy-driven malate cycling via the malate-aspartate shuttle, resulting in a lowered redox segregation of the NAD systems between the mitochondrial and extramitochondrial spaces. If pyruvate carboxylation was blocked, this egress of reducing equivalents was also blocked, leading to an elevated value of redox segregation, delta G(redox) (in kJ) = -5.7 log(NAD+/NADHout)/(NAD+/NADHin) being then equal to approximately one-half of the membrane potential, in accordance with electrogenic glutamate/aspartate exchange. Reconstitution of malate-pyruvate cycling led to a further kinetic decrease in the original malate-aspartate shuttle-driven value of delta G(redox). Therefore, the value of segregation of reducing potential between mitochondria and cytosol caused by glutamate/aspartate exchange can be diminished kinetically by processes exporting reducing equivalents from mitochondria, such as pyruvate carboxylation and pyruvate cycling.
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PMID:Control of reversible intracellular transfer of reducing potential. 182 12


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