Photoaffinity Labeling of the ATP Transporter of Rat Liver Microsomes

Seung Hyun Kim, Seung Jin Shin, Jae Chul Pyun, Ji Ho Yoo, Jong Sang Park

Research output: Contribution to journalArticle

Abstract

A photoreactive azido derivative of ATP, 3 (2′)-O-(p-azidobenzoyl)-ATP (AB-ATP), was synthesized by the reaction of ATP with N-hydroxysuccinimidyl-4-azidobenzoate (NHS-AB) to photolabel the ATP transporter of rat liver microsomes. AB-ATP specifically inhibited the transport of ATP into microsomes, 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 microsomes. An SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of microsomes photolabeled with [γ-32P]AB-ATP indicates the presence of four major protein bands with apparent molecular sizes of 97, 56, 53, and 47 kDa. Among these labeled proteins, the 56 kDa protein was completely protected from the photoaffinity labeling by 30 μM ATP but not by the same amount of GTP, which is consistent with the specific labeling of the ATP binding site of the ATP transporter. The specific labeling of the only 56 kDa protein among them was sensitive to the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) but not sensitive to the mitochondrial ADP/ATP carrier inhibitor, atractyloside (ATR). Moreover, the 56 kDa protein was uniquely photolabeled with [γ-32P]AB-ATP in the highly purified rough endoplasmic reticulum (RER) vesicles. These results strongly suggested that the 56 kDa protein represents the ATP transporter of rat liver endoplasmic reticulum (ER).

Original languageEnglish
Pages (from-to)582-588
Number of pages7
JournalMolecules and cells
Volume7
Issue number5
Publication statusPublished - 1997 Oct 31

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Photoaffinity Labeling of the ATP Transporter of Rat Liver Microsomes'. Together they form a unique fingerprint.

  • Cite this

    Kim, S. H., Shin, S. J., Pyun, J. C., Yoo, J. H., & Park, J. S. (1997). Photoaffinity Labeling of the ATP Transporter of Rat Liver Microsomes. Molecules and cells, 7(5), 582-588.