Sulfhydryl groups of the uncoupling protein of brown adipose tissue mitochondria.

Mersalyl, 5,5′-dithio-bis(2-nitrobenzoate) (Nbs₂) and fluorescent Thiolyte DB react with SH groups in the H⁺ channel (SH_C) of the uncoupling protein of brown adipose tissue mitochondria, as inferred from their inhibition of H⁺ transport. C^- transport by the uncoupling protein was unaffected. Using these modifiers and N-ethylmaleimide (MalNEt), distinct SH groups (SH_B) in the purine nucleotide binding site were identified. Nbs₂ reacts more readily with the SH_B than with the SH_C groups, but mersalyl and Thiolyte DB are more reactive with the SH_C groups. MalNEt reacts exclusively with the SHB. GDP inhibition is fully prevented after sufficient modification of the SH_B. Pretreatment with p-diazobenzenesulfonate (N₂PhSO₂) suppresses only 20–25% of fluorescence of Thiolyte-DB-labeled uncoupling protein on SDS/PAGE gels, while MalNEt suppresses 66% and Nbs₂ 80–90%. Since N₂PhSO₂ also affects the GDP binding site, these results demonstrate that the reactive residue is not identical with the SHB. [ABSTRACT FROM AUTHOR]