Factors affecting mitochondrial respiration in yeast Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/h989r552s

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  • Two experimental approaches have been used to investigate the interaction of sterols with respiration in yeast. Cells were tested with an antifungal sterol, azasterol, and a mutant was obtained with an altered sterol composition. Polarographic measurements were done on mitochondria for both experiments. The effects of azasterol on mitochondrial respiration were tested in two ways. In the first experiment, azasterol was added directly to mitochondria. The second method involved the isolation of mitochondria from azasterol grown cells. In the former, polarographic measurements with ethanol as substrate showed no coupling of mitochondria in the range of 25-50 μg azasterol/ml. Mitochondria from cells grown in the presence of azasterol were inhibited at much lower concentrations. No respiration was observed at 25 ng azasterol/ml, and respiration with no coupling occurred at 10 ng azasterol/ml. Coupling wμs present at 1.25 ng azasterol/ml. All three azasterol concentrations showed similar ignosterol/ergosterol ratios by U.V. absorption, suggesting ignosterol may not be the only factor affecting respiration. Polarographic measurements were made with a sterol mutant and wild-type mitochondria at varying temperatures. Coupling was observed with the mutant above the permissive growth temperature. The R/C and ATP/0 ratios were similar to the wild-type. The respiratory control ratios were measured by dividing the polarographic slope of state 3 respiration (mitochondria in the presence of substrate and ADP) by the polarographic slope of state 4 respiration (mitochondria in the presence of substrate and the ADP is utilized). However, the mutant was more susceptible to Na⁺ ions, suggesting altered permeability, and had higher Q0₂ values at the elevated temperatures. The Q0₂ values suggest the sterol is not as effective in forming the intermediate gel state at the elevated temperatures as ergosterol. Results from the two experiments suggest there is an interaction between sterols and respiration.
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