Photoaffinity labeling of a synaptic vesicle specific nucleotide transport system from Torpedo marmorata

Abstract

We have employed azido derivatives of ATP and AMP to identify the ATP translocase of synaptic vesicles. Azido−AMP inhibits transport of both ATP and AMP in vitro. The affinity of the translocase for the azido derivatives is similar to that of the native ligands. Upon UV irradiation of vesicles incubated with radiolabeled azido−AMP or −ATP, a molecular weight (Mr) 34000 polypeptide is selectively modified. On two−dimensional gel electrophoresis, the single radiolabeled polypeptide has a pI of approximately 7.7. Analysis of the fractions obtained when vesicles were purified on linear sucrose density gradients reveals that the Mr 34000 polypeptide is highly enriched in the vesicle−containing fractions. The findings support the notion that this polypeptide is identical with a previously described vesicle−specific component of the same molecular size [Stadler, H., & Tashiro, T. (1979) Eur. J. Biochem. 101, 171−178], and we conclude on the basis of uptake inhibition and photoaffinity labeling results that this protein is directly involved in ATP translocation of synaptic vesicle