Tommasino M, Prezioso G, Palmieri F
Biochim Biophys Acta. 1987 Jan 16;890(1):39-46. doi: 10.1016/0005-2728(87)90066-1.
The effect of 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive analogue of phosphate, on the phosphate carrier of pig-heart mitochondria has been investigated. In the dark, ANPP inhibits the transport of phosphate in a competitive manner with a Ki of 3.2 mM. Upon photoirradiation with visible light, [32P]ANPP binds covalently to the phosphate carrier and the inhibition becomes irreversible. Both the inhibition of phosphate transport and the incorporation of [32P]ANPP into the phosphate carrier depend on the concentration of the inhibitor and the pH of the medium. Incubation of the mitochondria with phosphate during illumination in the presence of ANPP protects the carrier against inactivation and decreases the amount of radioactivity which is found to be associated with the purified protein. By extrapolation it is calculated that at 100% inactivation of the phosphate carrier 0.35 mol of reagent are bound per mol of 33 kDa carrier protein. It is concluded that ANPP can be used for photoaffinity labeling of the mitochondrial phosphate carrier at the substrate-binding site.
已对磷酸的光反应类似物4-叠氮基-2-硝基苯磷酸酯（ANPP）对猪心线粒体磷酸载体的作用进行了研究。在黑暗中，ANPP以竞争性方式抑制磷酸转运，其抑制常数Ki为3.2 mM。用可见光进行光照射时，[32P]ANPP与磷酸载体共价结合，抑制作用变得不可逆。磷酸转运的抑制和[32P]ANPP掺入磷酸载体均取决于抑制剂的浓度和介质的pH值。在ANPP存在下光照期间将线粒体与磷酸盐一起温育可保护载体不被失活，并减少与纯化蛋白相关的放射性量。通过外推法计算得出，在磷酸载体100%失活时，每摩尔33 kDa载体蛋白结合0.35摩尔试剂。得出的结论是，ANPP可用于在线粒体磷酸载体的底物结合位点进行光亲和标记。
