Klingenberg M, Grebe K, Scherer B
Eur J Biochem. 1975 Mar 17;52(2):351-63. doi: 10.1111/j.1432-1033.1975.tb04003.x.
35S-labelled atractylate and carboxy-atractylate are produced biosynthetically and used for studying the binding of these specific ligands to the ADP, ATP carrier in beef heart mitochondria. The following results are obtained. 1. Inhibition of translocation activity goes parallel to the increase of binding by [35S]atractylate. No additional binding is observed after full inhibition of translocation is reached giving evidence that atractylate binds exclusively to the carrier. 2. The maximum number of binding sites of both atractylates is about 1.6 mumol/g protein in beef heart mitochondria and decreases on treatment of the membrane by Pi, freezing, ageing, etc. The dissociation constants of the binding are approximately for atractylate Kd = 5-10(-8) M and for carboxy-atractylate Kd = 10(-8) M. The mass action plots of the concentration dependence for the binding are nonlinear-convex in particular with carboxy-atractylate and more linear with atractylate. Nonlinearity appears to be caused by some retardation of equilibration in the case of very high affinity binding. 3. The binding of atractylate and carboxy-atractylate is relatively fast in intact mitochondria and slower in aged membranes. There is a slower and a faster binding portion. 4. The atractylates remove ADP in a nearly 1:1 stoichiometry from untreated mitochondria. In aged and Pi-treated membranes the ratio deltaADP/deltaatractylate approaches 0. Obviously binding of carrier sites to ADP is more sensitive to alterations than that of the atractylates. The assumption is maintained that the binding site for atractylate is identical with that for ADP and ATP. 5. Bongkrekate prevents binding of both atractylates. However, when added after, it only removes atractylate but not the carboxy compound because of its different tight binding. The removal of atractylate depends on the synergistic effect of bongkrekate with ADP. 6. The binding studies with [35S]atractylate and in particular the interaction with bongkrekate support the reorienting carrier model in which atractylate as an impermeable ligand fixes the binding site of the carrier outside while with bongkrekate the carrier site is turned to the inside.
35S 标记的苍术酮酸和羧基苍术酮酸通过生物合成产生，并用于研究这些特定配体与牛心线粒体中 ADP、ATP 载体的结合。得到以下结果。1. 转运活性的抑制与[35S]苍术酮酸结合的增加平行。在转运完全被抑制后未观察到额外的结合，这表明苍术酮酸仅与载体结合。2. 两种苍术酮酸的最大结合位点数在牛心线粒体中约为 1.6 μmol/g 蛋白质，并且在经 Pi 处理、冷冻、老化等处理的膜上会减少。结合的解离常数对于苍术酮酸 Kd = 5×10⁻⁸ M，对于羧基苍术酮酸 Kd = 10⁻⁸ M。结合浓度依赖性的质量作用图是非线性凸形的，特别是对于羧基苍术酮酸，而对于苍术酮酸则更线性。非线性似乎是由极高亲和力结合情况下平衡的某种延迟引起的。3. 苍术酮酸和羧基苍术酮酸在完整线粒体中的结合相对较快，而在老化膜中较慢。存在较慢和较快的结合部分。4. 苍术酮酸以接近 1:1 的化学计量从未经处理的线粒体中去除 ADP。在老化和经 Pi 处理的膜中，ΔADP/Δ苍术酮酸的比值接近 0。显然，载体位点与 ADP 的结合比苍术酮酸的结合对变化更敏感。维持这样的假设，即苍术酮酸的结合位点与 ADP 和 ATP 的结合位点相同。5. 邦克里酸可防止两种苍术酮酸的结合。然而，当在之后添加时，由于其不同的紧密结合，它仅去除苍术酮酸而不去除羧基化合物。苍术酮酸的去除取决于邦克里酸与 ADP 的协同作用。6. 用[35S]苍术酮酸进行的结合研究，特别是与邦克里酸的相互作用，支持了重新定向载体模型，其中苍术酮酸作为一种不可渗透的配体将载体的结合位点固定在外部，而对于邦克里酸，载体位点转向内部。
