The F1Fo-ATPase Complex from Bovine Heart Mitochondria:  The Molar Ratio of the Subunits in the Stalk Region Linking the F1and FoDomains

Biochemistry ◽  
1996 ◽  
Vol 35 (38) ◽  
pp. 12640-12646 ◽  
Author(s):  
Ian R. Collinson ◽  
J. Mark Skehel ◽  
Ian M. Fearnley ◽  
Michael J. Runswick ◽  
John E. Walker
Keyword(s):  
Heart Mitochondria ◽  
Molar Ratio ◽  
Bovine Heart ◽  
F1fo Atpase

scholarly journals Large-scale chromatographic purification of F1F0-ATPase and complex I from bovine heart mitochondria

1996 ◽  
Vol 318 (1) ◽  
pp. 343-349 ◽  
Author(s):  
Susan K BUCHANAN ◽  
John E. WALKER
Keyword(s):  
Complex I ◽  
Large Scale ◽  
Gel Filtration ◽  
Atp Synthesis ◽  
Lipid Vesicles ◽  
Proton Pumping ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Bovine Heart ◽  
F1fo Atpase

A new chromatographic procedure has been developed for the isolation of F1Fo-ATPase and NADH:ubiquinone oxidoreductase (complex I) from a single batch of bovine heart mitochondria. The method employed dodecyl β-Δ-maltoside, a monodisperse, homogeneous detergent in which many respiratory complexes exhibit high activity, for solubilization and subsequent purification by ammonium sulphate fractionation and column chromatography. A combination of anion-exchange, gel-filtration, and dye-ligand affinity chromatography was used to purify both complexes to homogeneity. The F1Fo-ATPase preparation contains only the 16 known subunits of the enzyme. It has oligomycin-sensitive ATP hydrolysis activity and, as demonstrated elsewhere, when reconstituted into lipid vesicles it is capable of ATP-dependent proton pumping and of ATP synthesis driven by a proton gradient [Groth and Walker (1996) Biochem. J. 318, 351–357]. The complex I preparation contains all of the subunits identified in other preparations of the enzyme, and has rotenone-sensitive NADH:ubiquinone oxidoreductase and NADH:ferricyanide oxidoreductase activities. The procedure is rapid and reproducible, yielding 50–80 mg of purified F1Fo-ATPase and 20–40 mg of purified complex I from 1 g of mitochondrial membranes. Both preparations are devoid of phospholipids, and gel filtration and dynamic light scattering experiments indicate that they are monodisperse. Therefore, the preparations fulfil important prerequisites for structural analysis.

scholarly journals ATP synthase from bovine heart mitochondria: reconstitution into unilamellar phospholipid vesicles of the pure enzyme in a functional state

1996 ◽  
Vol 318 (1) ◽  
pp. 351-357 ◽  
Author(s):  
Georg GROTH ◽  
John E. WALKER
Keyword(s):  
Atp Hydrolysis ◽  
Three Dimensional ◽  
Biochemical Properties ◽  
Proton Gradient ◽  
Heart Mitochondria ◽  
Phospholipid Vesicles ◽  
Dependent Manner ◽  
Biological Functions ◽  
Bovine Heart ◽  
F1fo Atpase

A highly purified and monodisperse preparation of proton-translocating F1Fo-ATPase from bovine heart mitochondria is an assembly of 16 unlike polypeptides. This preparation has been reconstituted in the presence of various detergents into unilamellar phospholipid vesicles. Incorporation of the enzyme into vesicles increases the ATP hydrolase activity of the enzyme by 10–20-fold, depending on the detergent, and the highest activities of ATP hydrolysis, 70 units/mg, were obtained by reconstitution from dodecylmaltoside or CHAPS. This activity is mostly sensitive to inhibitors that act on the Fo membrane sector of the complex. From the quenching of the pH-sensitive probe, 9-amino-6-chloro-2-methoxyacridine, it was shown that the reconstituted enzyme was able to form a transmembrane proton gradient in an ATP-dependent manner. By co-reconstitution of the enzyme with bacteriorhodopsin, it was demonstrated that in the presence of a light-induced proton gradient the enzyme can synthesize ATP from ADP and phosphate. Therefore, the characteristic biological functions of the F1Fo-ATPase in mitochondria have been demonstrated with the purified enzyme. Thus, in terms of both its physical and biochemical properties, the purified enzyme fulfils important pre-requisites for formation of two- and three-dimensional crystals.

scholarly journals The structure and subunit composition of the particulate NADH-ubiquinone reductase of bovine heart mitochondria

1976 ◽  
Vol 154 (2) ◽  
pp. 295-305 ◽  
Author(s):  
C. I Ragan
Keyword(s):  
Molecular Weight ◽  
Complex I ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Water Soluble ◽  
Heart Mitochondria ◽  
Molar Ratio ◽  
Polypeptide Composition ◽  
Molecular Weights ◽  
Bovine Heart

Preparations of NADH-ubiquinone reductase from bovine heart mitochondria (Complex I) were shown to contain at least 16 polypeptides by gel electrophoresis in the presence of sodium dodecyl sulphate. 2. High-molecular-weight soluble NADH dehydrogenase prepared from Triton X-100 extracts of submitochondrial particles [Baugh & King (1972) Biochem. Biophys. Res. Commun. 49, 1165-1173] was similar to Complex I in its polypeptide composition. 3. Solubilization of Complex I by phospholipase A treatment and subsequent sucrose-density-gradient centrifugation did not alter the polypeptide composition. 4. Lysophosphatidylcholine treatment of Complex I caused some selective solubilization of a polypeptide of mol.wt. 33000 previosuly postulated to be the transmembrane component of Complex I in the mitochondrial membrane [Ragan (1975) in Energy Transducing Membranes: Structure, Function and Reconstitution (Bennun, Bacila & Najjar, eds.), Junk, The Hague, in the press]. 5. Chaotropic resolution of Complex I caused solubilization of polypeptides of molecular weights 75000, 53000, 29000, 26000 and 15500 and traces of others in the 10000-20000-mol.wt.range. 6. The major components of the iron-protein fraction from chaotropic resolution had molecular weights of 75000, 53000 and 29000, whereas the flavoprotein contained polypeptides of molecular weights 53000 and 26000 in a 1:1 molar ratio. 7. Iodination of Complex I by lactoperoxidase indicated that the water-soluble polypeptides released by chaotropic resolution, in particular those of the flavoprotein fraction, were largely buried in the intact Complex. 8. The polypeptides of molecular weights 75000, 53000, 42000, 39000, 33000, 29000 and 26000 were present in 1:2:1:1:1:1:1 molar proportions. The two subunits of molecular weight 53000 are probably non-identical.

scholarly journals Purification and Characteristics of a Butyryl Coenzyme A Synthetase from Bovine Heart Mitochondria

1965 ◽  
Vol 240 (1) ◽  
pp. 29-33
Author(s):  
Leslie T. Webster ◽  
Lance D. Gerowin ◽  
Louis Rakita
Keyword(s):  
Coenzyme A ◽  
Heart Mitochondria ◽  
Bovine Heart

scholarly journals ATP Synthase Complex from Bovine Heart Mitochondria

1989 ◽  
Vol 264 (26) ◽  
pp. 15548-15551
Author(s):  
S Joshi ◽  
M J Pringle
Keyword(s):  
Atp Synthase ◽  
Heart Mitochondria ◽  
Bovine Heart

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

1975 ◽  
Vol 148 (3) ◽  
pp. 533-537 ◽  
Author(s):  
R B Beechey ◽  
S A Hubbard ◽  
P E Linnett ◽  
A D Mitchell ◽  
E A Munn
Keyword(s):  
Electron Microscopy ◽  
Rapid Method ◽  
Adenosine Triphosphatase ◽  
Pure Form ◽  
Heart Mitochondria ◽  
Polyacrylamide Gels ◽  
Submitochondrial Particles ◽  
Bovine Heart

An almost pure form of the bovine heart mitochondrial adenosine triphosphatase (ATPase) is released from the membrane by shaking submitochondrial particles with chloroform. Analyses on polyacrylamide gels and by electron microscopy, and also sensitivity to inhibitors, show that the chloroform-released enzyme is similar to other ATPase preparations from bovine heart mitochondria.

Proton flow through the intramembrane sector of the ATP synthase complex of bovine heart mitochondria

1987 ◽  
Vol 17 (1) ◽  
pp. 147-152
Author(s):  
G. Lippe ◽  
A. Perardi ◽  
M.C. Sorgato ◽  
F. Dabbeni-Sala
Keyword(s):  
Atp Synthase ◽  
Heart Mitochondria ◽  
Bovine Heart ◽  
Flow Through

scholarly journals NADH:ubiquinone oxidoreductase from bovine heart mitochondria Complementary DNA sequence of the import precursor of the 10 kDa subunit of the flavoprotein fragment

FEBS Letters ◽  
1991 ◽  
Vol 282 (1) ◽  
pp. 135-138 ◽  
Author(s):  
J.Mark Skehel ◽  
Stephanie J. Pilkington ◽  
Michael J. Runswick ◽  
Ian M. Fearnley ◽  
John E. Walker
Keyword(s):  
Dna Sequence ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Complementary Dna ◽  
Bovine Heart
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