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

Biochemistry
S H KimJ Park

Abstract

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...Continue Reading

Citations

May 1, 2007·Biochimica Et Biophysica Acta·Miklós CsalaGábor Bánhegyi
Dec 22, 1999·Seminars in Cell & Developmental Biology·F J Stevens, Y Argon
Oct 20, 2018·Biological Reviews of the Cambridge Philosophical Society·Maria R DepaoliRoland Malli
Aug 2, 2012·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Yuji IshiiHideyuki Yamada

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