Characterization of cysteine residues of mitochondrial ADP/ATP carrier with the SH-reagents eosin 5-maleimide and N-ethylmaleimide.

The effects of the membrane-impermeable fluorescent sulfhydryl reagent eosin 5-maleimide (EMA) and the membrane-permeable sulfhydryl reagent N-ethylmaleimide (NEM) on ADP transport via the ADP/ATP carrier and their labeling sites in the carrier were studied in bovine heart submitochondrial particles. Of the 4 cysteine residues in the carrier, EMA labeled Cys159 very rapidly, Cys56 slowly, and Cys256 very slowly and did not label Cys128. Its labeling of Cys159 was associated with inhibition of the ADP transport, suggesting that the peptide segment containing Cys159 is involved in the transport of adenine nucleotides. In contrast to the very rapid binding of EMA to Cys159, NEM bound to Cys56 more slowly and labeled Cys159 and Cys256 much more slowly. Like EMA, it did not react with Cys128. The labeling of Cys56 with NEM also inhibited ADP transport. From these results, the locations of these cysteine residues in the ADP/ATP carrier are discussed in relation to the transport of adenine nucleotides mediated by the ADP/ATP carrier.