Bammel B P, Brand J A, Germon W, Smith J C
Arch Biochem Biophys. 1986 Jan;244(1):67-84. doi: 10.1016/0003-9861(86)90095-0.
Some aspects of the interaction of the extrinsic, potential-sensitive, molecular probe diS-C3-(5) with pigeon heart mitochondria are reported in this paper. Binding studies based on fluorimetry indicate that the ratio of the dissociation constant to the maximum number of binding sites, KD/n, is larger for succinate-containing mitochondria than that for cyanide-inhibited preparations. These observations suggest that the basis of the energy-dependent diS-C3-(5) optical signals is the ejection of the probe from the mitochondrial membrane. A more detailed analysis indicated that the major change in the binding parameters is a reduction in the maximum number of binding sites, n, when a charge gradient is formed at the expense of substrate. Using rapid mixing techniques, the time course of the passive association of diS-C3-(5) with mitochondria, that of the glutamate- and ATP-dependent optical signals, and the effect of this probe on the rate at which the energy-dependent cytochrome c oxidase Soret band shift signal develops have been monitored. Retardation the ATP-dependent cytochrome c oxidase Soret band shift signal suggests that the probe readily permeates the mitochondrial membrane. The first-order rate law that the glutamate-dependent signal obeys suggests that the rate-limiting step in the development of this signal is the dissociation of the dye from the mitochondrial membrane or the permeation of this membrane by the probe. The faster phase of the ATP-induced signal likely reflects the initial transfer of dye from the bulk aqueous phase followed by a slower probe permeation process that obeys a first-order rate law. This probe appears to distribute across the mitochondrial membrane in accordance with the transmembrane potential as judged by its effect on the ATP-dependent cytochrome c oxidase Soret band shift signal. DiS-C3-(5) also appears to inhibit the NADH dehydrogenase.
本文报道了外在的、电位敏感的分子探针二硫代氰基罗丹明C3-(5)与鸽心线粒体相互作用的某些方面。基于荧光法的结合研究表明，对于含琥珀酸的线粒体，解离常数与最大结合位点数之比KD/n大于氰化物抑制制剂的该比值。这些观察结果表明，能量依赖性二硫代氰基罗丹明C3-(5)光学信号的基础是探针从线粒体膜中排出。更详细的分析表明，当以底物为代价形成电荷梯度时，结合参数的主要变化是最大结合位点数n的减少。使用快速混合技术，监测了二硫代氰基罗丹明C3-(5)与线粒体被动结合的时间进程、谷氨酸和ATP依赖性光学信号的时间进程，以及该探针对能量依赖性细胞色素c氧化酶Soret带位移信号发展速率的影响。ATP依赖性细胞色素c氧化酶Soret带位移信号的延迟表明该探针易于渗透线粒体膜。谷氨酸依赖性信号遵循的一级速率定律表明，该信号发展的限速步骤是染料从线粒体膜上解离或探针渗透该膜。ATP诱导信号的较快阶段可能反映了染料从本体水相的初始转移，随后是一个较慢的探针渗透过程，该过程遵循一级速率定律。根据其对ATP依赖性细胞色素c氧化酶Soret带位移信号的影响判断，该探针似乎根据跨膜电位分布在线粒体膜上。二硫代氰基罗丹明C3-(5)似乎也抑制NADH脱氢酶。
