Selective labeling and rotational diffusion of the ADP/ATP translocator in the inner mitochondrial membrane.

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.