The role of thiols in nucleotide uptake into synaptic vesicles from Torpedo marmorata

We have employed sulfhydryl group reagents in an attempt to determine the mechanism by which the transport of nucleotides into synaptic vesicles is controlled. Transport proved to be sensitive to N-ethylmaleimide; radiolabelled N-ethylmaleimide was used to locate the sulfhydryl group to the translocase-associated molecule previously identified as a polypeptide of Mr 34,000 [Lee and Witzemann (1983) Biochemistry 22, 6123-6130]. The nucleotide uptake was 75% inhibited by the mercurials rho-hydroxymercuribenzoate and rho-chloromercuriphenylsulfonate. Uptake was also sensitive to the reagents phenylarsine oxide and iodosobenzoic acid, which are specific for dithiols. These results indicate that a readily accessible dithiol is critical for nucleotide transport. Using the lipophilic oxidants iodosobenzoic acid and plumbagin, we demonstrated that nucleotide uptake was inhibited upon oxidation of the dithiol but that this did not involve an alteration in the affinity of the translocase for its substrate.