Oxidative Phosphorylation Coupled to Respiration in Highly Purified Intact Euglena Mitochondria
| Autor: | Tekchand Saidha, Benito Gómez-Silva, Arthur I. Stern, Jerome A. Schiff |
|---|---|
| Rok vydání: | 1985 |
| Předmět: |
Differential centrifugation
Euglena gracilis Physiology ved/biology Cellular respiration ved/biology.organism_classification_rank.species Plant Science Oxidative phosphorylation Antimycin A Biology biology.organism_classification Euglena chemistry.chemical_compound Biochemistry chemistry Dinitrophenol Agronomy and Crop Science Percoll |
| Zdroj: | Journal of Plant Physiology. 120:431-440 |
| ISSN: | 0176-1617 |
| DOI: | 10.1016/s0176-1617(85)80152-8 |
| Popis: | Summary Improved methods for isolation of mitochondria from Euglena gracilis var. bacillaris are presented based on Yeda press breakage of protease-prepared sphaeroplasts of low vitamin B12 grown cells. Active preparations are obtained from dark-grown cells of wild-type or mutants W3BUL or W10BSmL but W1o is preferred since it lacks proplastids that can contaminate the mitochondrial preparations. Crude mitochondria obtained by differential centrifugation are further purified on Percoll gradients yielding five fractions (in order of decreasing density): A) enriched in clean contracted mitochondria; B) enriched in clean orthodox mitochondria; and C) D) and E) which contain broken mitochondria and mitochondrial membrane vesicles. All fractions show succinic dehydrogenase activity but only fractions A and B are intact based on retention of fumarase activity. Fraction A contains the bulk of the protein and consistently shows the highest respiratory activity, the activity of fraction B is somewhat lower and resembles the activity of crude mitochondria; fractions C, D and E are inactive. Oxygen uptake (unstimulated by ADP) by fraction A mitochondria is inhibited 100 % by 0.1 mM KCN, about 30 % by 6.6 μM antimycin A and about 50 % by 5 mM NaN3. True uncoupling is observed for the first time in Euglena mitochondria since dinitrophenol (DNP) (0.16 mM) and carbonylcyanide mtrichlorophenylhydrazone (m-CCCP) (0.2 μM) increase O2 uptake by about 250 %. P to O ratios of 2 - 3 are obtained. Electron microscopy and plating on rich media indicate that these preparations are free of significant contamination with other cell fractions or with microorganisms. |
| Databáze: | OpenAIRE |
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