Lauquin G J, Vignais P V
Biochemistry. 1976 Jun 1;15(11):2316-22. doi: 10.1021/bi00656a011.
Chemical labeling by 3H and biosynthetic labeling by 14C of bongkrekic acid (BA) are described. In the rat liver cell, mitochondria are the only subcellular particles to bind [3H]BA with high affinity. The high affinity sites for BA in mitochondria are located in the inner membrane. High affinity binding sites for BA are only displayed at pH below 7; they amount to 0.15-0.20 nmol/mg of protein in rat liver mitochondria and to 1.1-1.3 nmol/mg of protein in rat heart mitochondria. These values are similar to those found for the high affinity atractyloside binding sites and for the carboxyatractyloside binding sites. The kinetic parameters for BA binding to rat heart mitochondria at 20 degrees C are Kd = 10-40 X 10(-9) M, k+1 = 0.7 X 10(5) M-1 s-1, k-1 = 1.4 X 10(-3) M s-1. Binding assays carried out with rat heart mitochondria, under equilibrium conditions, showed that the amount of BA bound to high affinity sites increases with temperature and reaches the maximum value of 1.1-1.3 nmol/mg of protein at 32-35 degrees C. At lower temperatures, and under equilibrium conditions, a significant fraction of high affinity sites remains masked and is not titrated by BA; these masked BA sites are revealed by addition of micromolar concentrations of ADP or by energization of the mitochondria. Carboxyatractyloside added to rat heart mitochondria preloaded with [3H]BA is able to displace part of the bound [3H]BA. Displacement of the bound BA is enhanced by simultaneous additions of carboxyatractyloside plus ADP, or by energization of the mitochondria. The synergistic effect of carboxyatractyloside and ADP on displacement of bound [3H]BA is also observed in isolated inner membrane vesicles from rat liver mitochondria. When BA is preincubated with rat heart mitochondria before addition of [14C]ADP for assay of ADP transport, the inhibition of ADP transport is a mixed-type inhibition. When BA is preincubated with the mitochondria together with a very small concentration of ADP (less than 0.5 muM), the inhibition of [14C]ADP transport is markedly increased (up to ten times) and it becomes typically uncompetitive, which suggests the formation of a ternary complex, carrier-ADP-BA. The transition from a mixed-type inhibition, with high Ki value, to an uncompetitive type of inhibition, with low Ki value, upon addition of ADP, is explained by an ADP-induced conformational change of the ADP translocator.
本文描述了用³H进行的化学标记以及用¹⁴C进行的邦克里尔酸（BA）的生物合成标记。在大鼠肝细胞中，线粒体是唯一能与[³H]BA高亲和力结合的亚细胞颗粒。线粒体中BA的高亲和力位点位于内膜。BA的高亲和力结合位点仅在pH低于7时显示；在大鼠肝脏线粒体中，它们的含量为0.15 - 0.20 nmol/mg蛋白质，在大鼠心脏线粒体中为1.1 - 1.3 nmol/mg蛋白质。这些值与在高亲和力的苍术苷结合位点和羧基苍术苷结合位点发现的值相似。20℃时BA与大鼠心脏线粒体结合的动力学参数为：Kd = 10 - 40×10⁻⁹M，k⁺¹ = 0.7×10⁵M⁻¹s⁻¹，k⁻¹ = 1.4×10⁻³M s⁻¹。在平衡条件下用大鼠心脏线粒体进行的结合测定表明，与高亲和力位点结合的BA量随温度升高而增加，在32 - 35℃时达到1.1 - 1.3 nmol/mg蛋白质的最大值。在较低温度和平衡条件下，相当一部分高亲和力位点仍被掩盖，未被BA滴定；加入微摩尔浓度的ADP或使线粒体 energization可揭示这些被掩盖的BA位点。加入预先用[³H]BA预加载的大鼠心脏线粒体中的羧基苍术苷能够取代部分结合的[³H]BA。同时加入羧基苍术苷和ADP或使线粒体energization可增强结合的BA的取代。在大鼠肝脏线粒体分离的内膜囊泡中也观察到羧基苍术苷和ADP对结合的[³H]BA取代的协同作用。当在加入[¹⁴C]ADP用于ADP转运测定之前将BA与大鼠心脏线粒体预孵育时，对ADP转运的抑制是混合型抑制。当将BA与线粒体一起用非常低浓度的ADP（小于0.5μM）预孵育时，对[¹⁴C]ADP转运的抑制明显增加（高达十倍），并且变得典型地为非竞争性抑制，这表明形成了三元复合物，载体 - ADP - BA。加入ADP后，从具有高Ki值的混合型抑制转变为具有低Ki值的非竞争性抑制类型，这是由ADP诱导的ADP转运体构象变化所解释的。 （注：energization这个词在生物学语境中可能有特定含义，但文中未明确解释，此处直接保留英文，需根据专业知识进一步理解其确切意义）
