Kim S H, Shin S J, Pyun J C, Yoo J H, Park J S
Department of Chemistry, Seoul National University, Korea.
Mol Cells. 1997 Oct 31;7(5):582-8.
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 [gamma-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 microM 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 [gamma-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).
通过ATP与N - 羟基琥珀酰亚胺 - 4 - 叠氮苯甲酸酯（NHS - AB）反应合成了一种ATP的光反应性叠氮衍生物，即3'（2'） - O - （对 - 叠氮苯甲酰） - ATP（AB - ATP），用于对大鼠肝微粒体的ATP转运体进行光标记。AB - ATP特异性抑制ATP转运至微粒体，这表明AB - ATP对ATP转运体具有高亲和力，因此它可作为光亲和探针用于鉴定大鼠肝微粒体中的ATP转运体。用[γ - 32P]AB - ATP进行光标记的微粒体的SDS - 聚丙烯酰胺凝胶电泳（SDS - PAGE）分析表明存在四条主要蛋白带，其表观分子大小分别为97、56、53和47 kDa。在这些标记蛋白中，56 kDa的蛋白可被30μM的ATP完全保护免于光亲和标记，但等量的GTP则不能，这与ATP转运体的ATP结合位点的特异性标记一致。其中仅56 kDa蛋白的特异性标记对阴离子转运抑制剂4,4'-二异硫氰酸 - 2,2'-二磺酸芪（DIDS）敏感，但对线粒体ADP/ATP载体抑制剂苍术苷（ATR）不敏感。此外，在高度纯化的糙面内质网（RER）小泡中，56 kDa的蛋白被[γ - 32P]AB - ATP独特地光标记。这些结果强烈表明56 kDa的蛋白代表大鼠肝内质网（ER）的ATP转运体。
