Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V
| Autor: | Shintaro Maeda, Shigefumi Uene, Kaoru Mieda-Higa, Masahide Hikita, Kyoko Shinzawa-Itoh, Satoru Shimada, Yukiko Nariai |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Potassium chemistry.chemical_element Mitochondrial Proton-Translocating ATPases Mitochondria Heart Mitochondrial Proteins 03 medical and health sciences chemistry.chemical_compound Electron Transport Complex III Animals Solubility Differential centrifugation Chromatography Electron Transport Complex I Chemistry Myocardium Deoxycholic acid 030104 developmental biology Membrane Mitochondrial respiratory chain Mitochondrial Membranes Cattle Biotechnology |
| Zdroj: | Protein expression and purification. 150 |
| ISSN: | 1096-0279 |
| Popis: | Ascertaining the structure and functions of mitochondrial respiratory chain complexes is essential to understanding the biological mechanisms of energy conversion; therefore, numerous studies have examined these complexes. A fundamental part of that research involves devising a method for purifying samples with good reproducibility; the samples obtained need to be stable and their constituents need to retain the same structure and functions they possess when in mitochondrial membranes. Submitochondrial bovine heart particles were isolated using differential centrifugation to adjust to a membrane concentration of 46.0% (w/v) or 31.5% (w/v) based on weight. After 0.7% (w/v) deoxycholic acid, 0.4% (w/v) decyl maltoside, and 7.2% (w/v) potassium chloride were added to the mitochondrial membranes, those membranes were solubilized. At a membrane concentration of 46%, complex V was selectively solubilized, whereas at a concentration of 31.5% (w/v), complexes I and III were solubilized. Two steps-sucrose density gradient centrifugation and anion-exchange chromatography on a POROS HQ 20 μm column-enabled selective purification of samples that retained their structure and functions. These two steps enabled complexes I, III, and V to be purified in two days with a high yield. Complexes I, III, and V were stabilized with n-decyl-β-D-maltoside. A total of 200 mg-300 mg of those complexes from one bovine heart (1.1 kg muscle) was purified with good reproducibility, and the complexes retained the same functions they possessed while in mitochondrial membranes. |
| Databáze: | OpenAIRE |
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