PHOTOAFFINITY-LABELING OF THE ATP TRANSPORTER OF RAT-LIVER MICROSOMES

A photoreactive azido derivative of ATP, 3 (2')-O-(p-azidobenzoyl)-ATP (AB-ATP), was synthesized by the reaction of ATP with N-hydroxysuccin imidyl-4-azidobenzoate (NHS-AB) to photolabel the ATP transporter of r at liver microsomes. AB-ATP specifically inhibited the transport of AT P into microsomes, which indicates that AB-ATP has a high affinity for the ATP transporter, so it can be utilized as a photoaffinity probe f or the identification of the ATP transporter in rat liver microsomes. An SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of micro somes photolabeled with [gamma-P-32]AB-ATP indicates the presence of f our major protein bands with apparent molecular sizes of 97, 56, 53, a nd 47 kDa. Among these labeled proteins, the 56 kDa protein was comple tely protected from the photoaffinity labeling by 30 mu M ATP but not by the same amount of GTP, which is consistent with the specific label ing of the ATP binding site of the ATP transporter. The specific label ing of the only 56 kDa protein among them was sensitive to the anion t ransport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) but not sensitive to the mitochondrial ADP/ATP carrier inhibito r, atractyloside (ATR). Moreover, the 56 kDa protein was uniquely phot olabeled with [gamma-P-32]AB-ATP in the highly purified rough endoplas mic reticulum (RER) vesicles. These results strongly suggested that th e 56 kDa protein represents the ATP transporter of rat liver endoplasm ic reticulum (ER).