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

1993; Elsevier BV; Volume: 268; Issue: 29 Linguagem: Inglês

10.1016/s0021-9258(20)80665-6

1083-351X

Eiji Majima, Haruhiko Koike, Yiyang Hong, Yasuo Shinohara, Hiroshi Terada,

Adipose Tissue and Metabolism

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 CyslSg very rapidly, CysS6 slowly, and very slowly and did not label CyslZs.Its labeling of CYS'~' was associated with inhibition of the ADP transport, suggesting that the peptide segment containing C ~S ' ' ~ is involved in the transport of adenine nucleotides.In contrast to the very rapid binding of EMA to C ~S ' ' ~, NEM bound to Cy$' more slowly and labeled CYS'~' and CysZ6' much more slowly.Like EMA, it did not react with C ~S ' ~~.The labeling of Cys" 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.