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Selective labeling and rotational diffusion of the ADP/ATP translocator in the inner mitochondrial membrane
Abstract:
Submitochondrial particles were labeled with the triplet probe eosin-5-maleimide (EMA) after pretreatment with N-ethylmaleimide. On sodium dodecyl sulfate-polyacrylamide gels, eosin fluorescence occurred in a single band of Mr approximately 30,000. The labeled band was identified as the ADP/ATP translocator, since EMA binding was completely inhibited by carboxyatractylate. Furthermore, the EMA-labeled polypeptide had the same molecular weight as the purified carboxyatractylate-bound translocator and the purified EMA-labeled translocator. Rotational diffusion of the translocator around the membrane normal in submitochondrial particles was measured by observing flash-induced absorption anisotropy of EMA. The translocator rotates with a time constant which varied from approximately 240 microseconds at 5 degrees C to approximately 100 microseconds at 37 degrees C. However, it is likely that only a fraction of the translocator rotates, the remainder being immobile over the measurement time of 500 microseconds. The mobile fraction of the translocator decreased with decrease in temperature. The observed fluorescence anisotropy of 0.24 indicates that EMA undergoes subnanosecond rapid wobbling in the binding site of the ADP/ATP translocator.
Insights
Submitochondrial particles revealed the ADP/ATP translocator using eosin-5-maleimide (EMA) labeling. This protein exhibits rotational diffusion within the membrane, with a temperature-dependent mobile fraction.
Area of Science:
- Mitochondrial biochemistry
- Membrane protein dynamics
- Biophysical techniques
Background:
- The ADP/ATP translocator is crucial for mitochondrial energy production.
- Understanding its membrane dynamics is key to elucidating mitochondrial function.
Purpose of the Study:
- To identify and characterize the rotational diffusion of the ADP/ATP translocator in submitochondrial particles.
- To investigate the temperature dependence of translocator mobility and its implications for function.
Main Methods:
- Submitochondrial particles were labeled with eosin-5-maleimide (EMA), a fluorescent probe.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for protein identification.
- Flash-induced absorption anisotropy measured the rotational diffusion of the EMA-labeled translocator.
Main Results:
- EMA specifically labeled the ADP/ATP translocator, confirmed by inhibition with carboxyatractylate.
- The translocator exhibited temperature-dependent rotational diffusion, with faster rotation at higher temperatures.
- A fraction of the translocator remained immobile, and EMA showed subnanosecond wobbling within its binding site.
Conclusions:
- The ADP/ATP translocator undergoes rotational diffusion in the mitochondrial membrane.
- Translocator mobility is influenced by temperature, suggesting dynamic regulation of its function.
- The findings provide insights into the biophysical properties of this essential mitochondrial protein.