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

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Abstract
Azido derivatives of ATP and AMP were used to identify the ATP translocase of synaptic vesicles [from the electromotor tissue of T. marmorata]. 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 MW 34,000 polypeptide is selectively modified. On 2-dimensional gel electrophoresis, the single radiolabeled polypeptide has a pI of .apprx. 7.7. Analysis of the fractions obtained when vesicles were purified on linear sucrose density gradients reveals that the MW 34,000 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. On the basis of uptake inhibition and photoaffinity labeling results that this protein is directly involved in ATP translocation of synaptic vesicles.