Two Examples of Selective Fluorescent Labeling of SH-Groups with Eosin-5-Maleimide: The ADP/ATP Translocator and the Cytochrome c Oxidase Subunit III of Bovine Heart Mitochondria

Abstract

The ADP/ATP translocator is located in the inner membrane of mitochondria, where it catalyzes the vectorial exchange between cytosolic ADP and matrix ATP, a key process in the cellular energy supply of aerobic organisms (for review see Klingenberg 1980, Vignais 1976, Vignais et al. 1982). In beef heart mitochondria, the translocator is the most abundant integral protein, being about 10% of the total mitochondrial protein. The molecular weight of the monomeric translocator is about 32,500. The translocator has been extracted and purified as a dimeric carboxyatractylate (CAT) or bongkrekic acid complex by using Triton X-100. A complete amino acid sequence of the translocator revealed that the monomeric protein contains 4 cysteins out of the 297 amino acids. Sulfydryl groups are essential for the nucleotide translocation, since their hindrance or modification causes inhibition of the process. At least one sulfydryl group is masked when CAT is bound to the translocator. Eosin-5-maleimide (EMA, Fig. 1) reacts specifically with SH-groups and at low concentration does not permeate the inner membrane. In the absence of ADP the SH-groups of the ADP/ATP translocator do not react with N-ethylmaleimide (NEM), whereas all the other SH-groups located on the cytosolic side of the inner membrane react with NEM. The difference in reactivity between EMA an and NEM was used as an experimental approach to label selectively the ADP/ATP translocator in mitochondria, mitoplasts and submitochondrial particles (Müller et al. 1982, 1984). The aim of the experiment is to show the selectivity of the labeling and the effect of CAT and mersalyl on it.