Kim S H, Shin S J, Park J S
Department of Chemistry, Seoul National University, Korea.
Biochemistry. 1996 Apr 30;35(17):5418-25. doi: 10.1021/bi950485h.
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 [8-14C]ATP. The ATP transport had an apparent Km value of 6.5 microM and a Vmax 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 Ki value of 0.19 microM, 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-32P]AB-ATP indicates the presence of a 56-kDa protein. The 56-kDa protein was completely protected from photoaffinity labeling by 10 microM ATP but not by 30 microM 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-32P]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.
