A simple and rapid method for the preparation of adenosine triphosphatase from submitochondrial particles

Beechey, R. B.; Hubbard, S A; Linnett, Paul E.; Mitchell, A. D.; Munn, E. A.

doi:10.1042/bj1480533

Cited by 267 publications

(97 citation statements)

References 32 publications

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“…Fig. 4 also shows the composition of C. fasciculata F,, isolated from mitochondrial vesicles by chloroform extraction (lane 4), see [24], bovine mitochondrial F, and FoF, (lanes 1,2) and spinach chloroplast F0F ~ (lane 3). We first addressed the identity of the proteins in the F, preparation.…”

Section: The Subunit Composition Of Fofi-atpasementioning

confidence: 99%

“…Mt vesicles from 1.1 x 101~ cells of cultured C. fasciculata were lysed by sonication (3 x 10 s) on ice in the presence of 1 mM ATP, followed by extraction with chloroform, as described by Beechey et al [24], to obtain the F~ part from the FoF1-ATPase. Although not completely pure, proteins of 50, 40, 33, 23 and 10 kDa, found to comprise purified F~-ATPase from C. fasciculata [25] were among the major protein components in this preparation (see Section 3).…”

Section: Purification Of the F1 Fraction From Fof T -A Tpasementioning

confidence: 99%

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Characterization of the respiratory chain from cultured Crithidia fasciculata

Speijer

Breek

Muijsers

et al. 1997

Molecular and Biochemical Parasitology

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Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplastids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia Jasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of FoF~-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial FoF~-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two Fo subunits (b and c) and all of the F~ subunits. The Fob subunit has a low degree of similarity to subunit b from other organisms. The Fl c~ subunit is extremely small making the fl subunit the largest F~ subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex II I) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V. © 1997 Elsevier Science B.V.

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Section: The Subunit Composition Of Fofi-atpasementioning

confidence: 99%

Section: Purification Of the F1 Fraction From Fof T -A Tpasementioning

confidence: 99%

Characterization of the respiratory chain from cultured Crithidia fasciculata

Speijer

Breek

Muijsers

et al. 1997

Molecular and Biochemical Parasitology

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“…Bovine heart submitochondrial particles prepared as described in [17] and suspended in 0.25 M sucrose, 4 mM ATP, 1 mM EDTA, 10 mM Tris/HCl pH 9.2 were incubated at a protein concentration of 30mg/ml at room temperature for 8 h. The pH was then adjusted to 8.0 with 1 M HCl and crude F, -ATPase was released from particles by chloroform treatment as described by Beechey et al [18]. The aqueous phase was centrifuged for 30 min at 105000 X g at 20°C and the resulting supernatant was brought to 50% saturation with saturated ammonium sulfate solution (pH 7).…”

Section: Preparation Of F-atpase and Removal Of Nucleotidesmentioning

confidence: 99%

Tryptophan phosphorescence as a structural probe of mitochondrial F₁‐ATPase ε‐subunit

Solaini

Baracca

Castelli

et al. 1993

European Journal of Biochemistry

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We report the detection of tryptophan phosphorescence emission from the sole residue in the Esubunit of the bovine heart mitochondrial F, -ATPase complex. The phosphorescence spectrum, intensity and decay kinetics have been measured over the temperature range 160-273 K. The fine structure in the phosphorescence spectrum at low temperature, with the 0-0 vibrational band centered at 411 nm, reveals the hydrophobic nature of the chromophore's environment. Both the large width of the 0-0 vibrational band and the heterogeneous decay kinetics in fluid solution emphasize the existence of multiple conformations of the &-subunit, structures which are rather stable as they do not interconvert in the millisecond time scale. Further, from the relatively long triplet lifetime at 273 K, it is possible to infer the existence of a tight, rigid core in the structure of the &-subunit. Under subunit-dissociating conditions (6 M urea), the spectrum at 160 K undergoes a slight blue shift but since the phosphorescence lifetime, at all temperatures, is similar or longer than in the absence of dissociant, we conclude that dissociation does not lead to solvent exposure of the tryptophanyl side-chain. This conclusion is supported by the results obtained at 273 K by dissociating F, in the presence of 0.3 M guanidine hydrochloride. Phosphorescence lifetimes indicate that 6 M

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“…All subsequent steps were carried out at room temperature. The ATPase was released from the membranes by solvent treatment [14] in the presence of 1 mM freshly prepared PMSF, except that dichloromethane was used as a solvent. The ATPase was purified from the dichloromethane-solubilised material after centrifugation at 100000×g for 40 min in a Beckman 40 rotor by the method of O'Rourke [10].…”

Section: Methodsmentioning

confidence: 99%

The plant mitochondrial F₁‐ATPase

Horak

Dunbar

et al. 1990

FEBS Letters

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The N-terminal amino acid sequence of the 20 kDa (3') subunit of the turnip (Brassica napus L.) mitochondrial F~-ATPase has been determined. Comparison of the sequence obtained with those of the e subunits of chloroplast CF~, E. coli F 1 and the ~ subunit of bovine F~ shows that the turnip 3' subunit is another member of this family of homologous proteins. The 3' subunit of sweet potato F~-ATPase [(1989) J. Biol. Chem. 264, 3183-3186] is very similar to the turnip sequence and thus can also be considered to belong to this family.

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A simple and rapid method for the preparation of adenosine triphosphatase from submitochondrial particles

Cited by 267 publications

References 32 publications

Characterization of the respiratory chain from cultured Crithidia fasciculata

Characterization of the respiratory chain from cultured Crithidia fasciculata

Tryptophan phosphorescence as a structural probe of mitochondrial F₁‐ATPase ε‐subunit

The plant mitochondrial F₁‐ATPase

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A simple and rapid method for the preparation of adenosine triphosphatase from submitochondrial particles

Cited by 267 publications

References 32 publications

Characterization of the respiratory chain from cultured Crithidia fasciculata

Characterization of the respiratory chain from cultured Crithidia fasciculata

Tryptophan phosphorescence as a structural probe of mitochondrial F1‐ATPase ε‐subunit

The plant mitochondrial F1‐ATPase

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Product

Resources

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Tryptophan phosphorescence as a structural probe of mitochondrial F₁‐ATPase ε‐subunit

The plant mitochondrial F₁‐ATPase