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Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue
Published on: March 23, 2015
Mitochondrial membrane permeabilization and superoxide production during apoptosis. A single-cell analysis
Heiko Düssmann1, Donat Kögel, Markus Rehm
1Interdisciplinary Center for Clinical Research (IZKF), Westphalian Wilhelms-University, D-48149 Münster, Germany.
The Journal of Biological Chemistry
|February 1, 2003
Summary
Mitochondria increase superoxide production during apoptosis after releasing proteins. Caspase inhibitors like z-VAD-fmk reduce this superoxide surge and show antioxidant effects.
Area of Science:
- Cell Biology
- Biochemistry
- Apoptosis Research
Background:
- The precise timing of mitochondrial events during apoptosis is unclear.
- The relationship between mitochondrial membrane permeabilization and reactive oxygen species (ROS) production needs clarification.
Purpose of the Study:
- To investigate the temporal link between cytochrome c release and superoxide production during apoptosis.
- To analyze superoxide production kinetics at the single-cell level in human breast carcinoma cells.
Main Methods:
- Utilized a cytochrome c-green fluorescent protein (cyt-c-GFP) fusion protein to track mitochondrial release.
- Measured superoxide production using the hydroethidine probe in single cells.
- Administered proapoptotic agents (staurosporine, TNF-alpha) and caspase inhibitors (z-VAD-fmk).
Main Results:
- Cytochrome c-GFP release occurred rapidly (<10 min) after proapoptotic agent treatment.
- Superoxide production significantly increased (>3-fold) after cytochrome c release.
- Caspase inhibition (z-VAD-fmk) reduced post-release superoxide levels and menadione-induced superoxide spikes.
Conclusions:
- Mitochondria elevate superoxide production immediately following the release of intermembrane proteins during apoptosis.
- The caspase inhibitor z-VAD-fmk exhibits antioxidant properties by mitigating ROS increase post-cytochrome c release.
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