Stipani I, Natuzzi D, Daddabbo L, Ritieni A, Randazzo G, Palmieri F
Department of Pharmaco-Biology, University of Bari, Italy.
Biochim Biophys Acta. 1995 Mar 22;1234(2):149-54. doi: 10.1016/0005-2736(94)00291-v.
The effect of azido-phthalonate, a photoreactive analogue of oxoglutarate, on the transport of oxoglutarate was investigated in proteoliposomes reconstituted with the purified oxoglutarate carrier. In the dark, azido-phthalonate inhibits the reconstituted oxoglutarate/oxoglutarate exchange in a competitive manner with a Ki of 0.38 mM. Upon photoirradiation, the inhibition of the oxoglutarate exchange by azido-phthalonate is not removed by passing the proteoliposomes through a Sephadex column. The light-induced inhibition of the oxoglutarate/oxoglutarate exchange activity by azido-phthalonate is time- and concentration-dependent. The kinetic analysis of transport inhibition by azido-phthalonate reveals that one molecule of this substrate analogue bound to the functional carrier molecule is responsible for complete inhibition of the carrier function. Azido-[3H]phthalonate binds to the oxoglutarate carrier covalently. Incubation of the proteoliposomes with oxoglutarate during photoirradiation in the presence of azido-phthalonate protects the carrier against inactivation and decreases the amount of radioactivity which is found to be associated with the carrier protein. It is concluded that azido-phthalonate can be used for photoaffinity labeling of the mitochondrial oxoglutarate carrier at the substrate-binding site.
在由纯化的α-酮戊二酸载体重构的蛋白脂质体中，研究了α-酮戊二酸的光反应类似物叠氮邻苯二甲酸酯对α-酮戊二酸转运的影响。在黑暗中，叠氮邻苯二甲酸酯以竞争性方式抑制重构的α-酮戊二酸/α-酮戊二酸交换，其抑制常数Ki为0.38 mM。光照后，通过葡聚糖凝胶柱处理蛋白脂质体并不能消除叠氮邻苯二甲酸酯对α-酮戊二酸交换的抑制作用。叠氮邻苯二甲酸酯对α-酮戊二酸/α-酮戊二酸交换活性的光诱导抑制具有时间和浓度依赖性。对叠氮邻苯二甲酸酯转运抑制的动力学分析表明，与功能载体分子结合的一分子这种底物类似物可完全抑制载体功能。叠氮-[3H]邻苯二甲酸酯与α-酮戊二酸载体共价结合。在叠氮邻苯二甲酸酯存在下光照期间，用α-酮戊二酸孵育蛋白脂质体可保护载体不被失活，并减少与载体蛋白相关的放射性。得出的结论是，叠氮邻苯二甲酸酯可用于在线粒体α-酮戊二酸载体的底物结合位点进行光亲和标记。
