scholarly journals NADH:ubiquinone oxidoreductase from bovine heart mitochondria. cDNA sequences of the import precursors of the nuclear-encoded 39 kDa and 42 kDa subunits

1991 ◽  
Vol 278 (3) ◽  
pp. 821-829 ◽  
Author(s):  
I M Fearnley ◽  
M Finel ◽  
J M Skehel ◽  
J E Walker
Keyword(s):  
Amino Acid ◽  
Protein Sequence ◽  
Complex I ◽  
Mitochondrial Gene ◽  
Amino Acid Sequences ◽  
Heart Mitochondria ◽  
Blue Dye ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Cdna Clones ◽  
Bovine Heart

The 39 kDa and 42 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria are nuclear-coded components of the hydrophobic protein fraction of the enzyme. Their amino acid sequences have been deduced from the sequences of overlapping cDNA clones. These clones were amplified from total bovine heart cDNA by means of the polymerase chain reaction, with the use of complex mixtures of oligonucleotide primers based upon fragments of protein sequence determined at the N-terminals of the proteins and at internal sites. The protein sequences of the 39 kDa and 42 kDa subunits are 345 and 320 amino acid residues long respectively, and their calculated molecular masses are 39,115 Da and 36,693 Da. Both proteins are predominantly hydrophilic, but each contains one or two hydrophobic segments that could possibly be folded into transmembrane alpha-helices. The bovine 39 kDa protein sequence is related to that of a 40 kDa subunit from complex I from Neurospora crassa mitochondria; otherwise, it is not related significantly to any known sequence, including redox proteins and two polypeptides involved in import of proteins into mitochondria, known as the mitochondrial processing peptidase and the processing-enhancing protein. Therefore the functions of the 39 kDa and 42 kDa subunits of complex I are unknown. The mitochondrial gene product, ND4, a hydrophobic component of complex I with an apparent molecular mass of about 39 kDa, has been identified in preparations of the enzyme. This subunit stains faintly with Coomassie Blue dye, and in many gel systems it is not resolved from the nuclearcoded 36 kDa subunit.

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 The δ-subunit of ATP synthase from bovine heart mitochondria. Complementary DNA sequence of its import precursor cloned with the aid of the polymerase chain reaction

1990 ◽  
Vol 266 (2) ◽  
pp. 421-426 ◽  
Author(s):  
M J Runswick ◽  
S M Medd ◽  
J E Walker
Keyword(s):  
Polymerase Chain Reaction ◽  
Atp Synthase ◽  
Protein Sequence ◽  
Heart Mitochondria ◽  
Cdna Clones ◽  
Mature Protein ◽  
Chain Reaction ◽  
Bovine Heart ◽  
Polymerase Chain ◽  
Delta Subunit

The delta-subunit of ATP synthase from bovine heart mitochondria is part of the extrinsic membrane domain, F1-ATPase. The mature protein is 146 amino acids in length and its function is obscure. It is encoded by a nuclear gene and is imported into the organelle. Two mixtures of oligonucleotides 17 bases long, designed on the basis of the known protein sequence, have been synthesized and employed as primers on bovine cDNA in the polymerase chain reaction. By this means a segment of bovine cDNA encoding part of the delta-subunit has been amplified, and this DNA segment has been employed to identify related cDNA clones in a library. These clones encode the mitochondrial import precursor of the delta-subunit; the protein sequence of the mature protein deduced from it is exactly the same as that determined earlier by direct sequence analysis. The clones have also been used to show that both the bovine and human genomes seem to contain a single gene for the delta-subunit.

scholarly journals Investigation of the mechanism of proton translocation by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria: does the enzyme operate by a Q-cycle mechanism?

2006 ◽  
Vol 400 (3) ◽  
pp. 541-550 ◽  
Author(s):  
Steven Sherwood ◽  
Judy Hirst
Keyword(s):  
Complex I ◽  
Active Sites ◽  
Proton Translocation ◽  
Energy Transduction ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Protonmotive Force ◽  
Bovine Heart ◽  
Quinone Reduction ◽  
Q Cycle

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the membrane-bound electron transport chain in mitochondria. It conserves energy, from the reduction of ubiquinone by NADH, as a protonmotive force across the inner membrane, but the mechanism of energy transduction is not known. The structure of the hydrophilic arm of thermophilic complex I supports the idea that proton translocation is driven at (or close to) the point of quinone reduction, rather than at the point of NADH oxidation, with a chain of iron–sulfur clusters transferring electrons between the two active sites. Here, we describe experiments to determine whether complex I, isolated from bovine heart mitochondria, operates via a Q-cycle mechanism analogous to that observed in the cytochrome bc1 complex. No evidence for the ‘reductant-induced oxidation’ of ubiquinol could be detected; therefore no support for a Q-cycle mechanism was obtained. Unexpectedly, in the presence of NADH, complex I inhibited by either rotenone or piericidin A was found to catalyse the exchange of redox states between different quinone and quinol species, providing a possible route for future investigations into the mechanism of energy transduction.

Steady-State Kinetics of the Reduction of Coenzyme Q Analogs by Complex I (NADH:Ubiquinone Oxidoreductase) in Bovine Heart Mitochondria and Submitochondrial Particles†

Biochemistry ◽  
1996 ◽  
Vol 35 (8) ◽  
pp. 2705-2716 ◽  
Author(s):  
Romana Fato ◽  
Ernesto Estornell ◽  
Salvatore Di Bernardo ◽  
Francesco Pallotti ◽  
Giovanna Parenti Castelli ◽  
...  
Keyword(s):  
Steady State ◽  
Complex I ◽  
Coenzyme Q ◽  
Heart Mitochondria ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Submitochondrial Particles ◽  
Bovine Heart ◽  
Steady State Kinetics ◽  
Kinetics Of
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