Identification of the ATP transporter of rat liver rough endoplasmic reticulum via photoaffinity labeling and partial purification

In order to identify the ATP transporter in rat liver rough endoplasmic reticulum (RER), a photoreactive azido derivative of ATP, 3'-O-(p-azidobenzoyl)-ATP (AB-ATP), was synthesized by the reaction of ATP with N-hydroxysuccinimido 4-azidobenzoate (NHS-AB). The activity of the ATP transporter was determined by measuring the influx of [8-C-14]ATP. The ATP transport had an apparent K-m value of 6.5 mu M and a V-max of 1 nmol min(-1) (mg of protein)(-1). The transport of ATP was specifically inhibited by AB-ATP and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Under a dim light, AB-ATP was a competitive inhibitor of the ATP transport with a K-i value of 0.19 mu M, which indicates that AB-ATP has a high affinity for the ATP transporter, so it can be utilized as a photoaffinity probe for the identification of the ATP transporter in rat liver RER. An SDS-PAGE analysis of RER vesicles photolabeled with [gamma-P-32]AB-ATP indicates the presence of a 56-kDa protein. The 56-kDa protein was completely protected from photoaffinity labeling by 10 mu M ATP but not by 30 mu M GTP. The specific labeling of the 56-kDa protein was sensitive to the anion transport inhibitor DIDS. In order to confirm whether the apparent uptake of ATP was due to the 56-kDa protein, the ATP transporter was partially purified through two successive ion-exchange chromatography steps (DEAE and Mono-S). The fraction showing the high activity of the ATP transporter also contained the 56-kDa protein photolabeled with [gamma-P-32]AB-ATP. On the basis of the photoaffinity labeling and reconstitution experiment, we conclude that the 56-kDa protein represents the ATP transporter in rat liver RER.