title: Purification of Active Respiratory Supercomplex from Bovine Heart Mitochondria Enables Functional Studies.
abstract: To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this end, we solubilized supercomplexes from bovine heart mitochondria with digitonin and then replaced digitonin with amphipol (A8-35), an amphiphilic polymer. Afterward, supercomplexes were separated from other complexes by sucrose density gradient centrifugation. Twenty-six grams of bovine myocardium yielded 3.2 mg of amphipol-stabilized supercomplex. The purified supercomplexes were analyzed based on their absorption spectra as well as Q10 (ubiquinone with ten isoprene units) and lipid assays. The supercomplex sample did not contain cytochrome c but did contain complexes I, III, and IV at a ratio of 1:2:1, 6 molecules of Q10, and 623 atoms of phosphorus. When cytochrome c was added, the supercomplex exhibited KCN-sensitive NADH oxidation; thus, the purified supercomplex was active. Reduced complex IV absorbs at 444 nm, so we measured the resonance Raman spectrum of the reduced amphipol-solubilized supercomplex and the mixture of amphipol-solubilized complexes I1, III2, and IV1 using an excitation wavelength of 441.6 nm, allowing measurement precision comparable with that obtained for complex IV alone. Use of the purified active sample provides insights into the effects of supercomplex formation.
authors.affiliation: From the Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamighori, Akoh, Hyogo, 678-1297, Japan, shinzawa@sci.u-hyogo.ac.jp.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Shinzawa-Itoh
authors.affiliation: From the Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamighori, Akoh, Hyogo, 678-1297, Japan.
authors.forename: Harunobu
authors.initials: H
authors.lastname: Shimomura
authors.affiliation: From the Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamighori, Akoh, Hyogo, 678-1297, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Yanagisawa
authors.affiliation: From the Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamighori, Akoh, Hyogo, 678-1297, Japan, Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan, and.
authors.forename: Satoru
authors.initials: S
authors.lastname: Shimada
authors.affiliation: From the Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamighori, Akoh, Hyogo, 678-1297, Japan.
authors.forename: Ryoko
authors.initials: R
authors.lastname: Takahashi
authors.affiliation: From the Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamighori, Akoh, Hyogo, 678-1297, Japan.
authors.forename: Marika
authors.initials: M
authors.lastname: Oosaki
authors.affiliation: From the Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamighori, Akoh, Hyogo, 678-1297, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ogura
authors.affiliation: From the Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1, Koto, Kamighori, Akoh, Hyogo, 678-1297, Japan, Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan, and Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tomitake
authors.initials: T
authors.lastname: Tsukihara
mesh_terms: D000818:Animals; D002417:Cattle; D004072:Digitonin / Q000737:chemistry*; D045222:Electron Transport Chain Complex Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009124:Muscle Proteins / Q000737:chemistry / Q000302:isolation & purification*; D009206:Myocardium / Q000201:enzymology*; D011108:Polymers / Q000737:chemistry*; D011437:Propylamines / Q000737:chemistry*
chemical_list: D045222:Electron Transport Chain Complex Proteins; D009124:Muscle Proteins; D011108:Polymers; D011437:Propylamines; C499017:amphipol A8-35; D004072:Digitonin
keywords: Raman spectroscopy; bioenergetics; complex I; complex III; cytochrome c oxidase (complex IV); membrane protein; protein purification; respiratory supercomplex