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KMID : 0578319970070050582
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Molecules and Cells
1997 Volume.7 No. 5 p.582 ~ p.588
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Photoaffinity Labeling of the ATP Transporter of Rat Liver microsomes
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Kim, Seung-hyun
Shin, Seung-jin/Pyun, Jae-chul/Yoo, Ji-ho/Park, Jong-sang
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Abstract
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A photoreactive azido derivtive of ATP, 3 (2´)-O-(p-azidobenzoyI)-ATP (AB-ATP), was synthesized by the reaction of ATP with N-hydroxysuccinimidyI-4-azidobenzoate (NHS-AB) to photolabel the ATP transporter of rat liver microsomes. AB-ATP specifically inhibitied 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^(-32)P]AB-ATP indicates the presence of four major protein bands with apparent molecular size of 97, 56, 53, and 47 kDa. Among these labeled proteins, the 56 kDa protein was completely protected from the photoaffinity labeling by 30 ㎛ ATP but not by the same amount of GTP, which is consistent with the specific labelling of the ATP binding site of the ATP trnasporter. The specific labeling of the only 56kDa protein among them was sensitive to the anlon 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^(-32)]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).
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