MEMBRANE STRUCTURE STUDIED WITH CATIONIC DYES .1. BINDING OF CATIONIC DYES TO SUBMITOCHONDRIAL PARTICLES AND QUESTION OF POLARITY OF ION-TRANSLOCATION MECHANISM
MEMBRANE STRUCTURE STUDIED WITH CATIONIC DYES .1. BINDING OF CATIONIC DYES TO SUBMITOCHONDRIAL PARTICLES AND QUESTION OF POLARITY OF ION-TRANSLOCATION MECHANISM
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DOI：
10.1111/j.1432-1033.1972.tb19718.x
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发表时间：
1972-01-01
期刊：
影响因子：
--
通讯作者：
COLONNA, R
中科院分区：
文献类型：
--
作者：
DELLANTONE, P;AZZONE, GF;COLONNA, R
阳离子染料与在KCl培养基中孵育的失能亚线粒体颗粒弱相互作用。粒子的能量化导致结合的显著增加。Scatchard图显示，在中性红和pH 7.0的情况下，结合位点为150-200 μmol/g蛋白，解离常数约为2.5 μM。在相同pH下吖啶橙子的情况下，结合位点的值约为50 μmol/g蛋白质，解离常数约为2 μM。阳离子染料与赋能颗粒的结合伴随着吸光度的大幅降低，这在加入解偶联剂时逆转。吸光度的变化在化学计量上依赖于ATP的利用，但与能量是由呼吸还是ATP提供无关。在一定的蛋白质/染料浓度比范围内，反应动力学呈现准一级动力学特征，吸光度的变化与颗粒的数量成正比，直至达到饱和。这允许使用吸光度变化的“终点”或使用连续变化的方法滴定膜的染料结合位点。通过这些程序发现的结合位点的数量与结合实验获得的结果一致。介质的pH值对染料-膜相互作用有显著影响。当pH值从6增加到8时，结合速率增加，解离常数降低，结合位点数减少，亲脂性阳离子nuperadine对阳离子染料的结合有明显的抑制作用，吸光度随之降低。该抑制为竞争型，aKi为20 μM。介质中存在电解质是高亲和力结合和吸光度变化所必需的。用蔗糖代替KCl导致这两种现象的消除，并就亚线粒体颗粒中离子易位机制的极性倒置问题进行了讨论。有人认为，线粒体的超声处理会导致“极性丧失”而不是“极性反转”。的相互作用的动力学表明，这可能是所需的染料与通电网站的相互作用的限制步骤。
Cationic dyes interact weakly with de‐energized submitochondrial particles incubated in a KCl medium. Energization of the particles results in a marked increase of binding. The Scatchard plots reveal, in the case of neutral red, and at pH 7.0, binding sites of 150–200 μmol per g protein with a dissociation constant of about 2.5 μM. In the case of acridine orange at the same pH, the value for binding sites is about 50 μmol per g protein with a dissociation constant of about 2 μM.The binding of the cationic dyes to the energized particles is accompanied by a large decrease of absorbance which is reversed on addition of uncouplers. The change of absorbance is stoichiometrically dependent on the utilization of ATP, but independent of whether energy is supplied from respiration or from ATP. Within a certain range of protein/dye concentration ratio the reaction kinetics appears of pseudo first‐order character.The change of absorbance is proportional to the amount of particles until a saturation is obtained. This permits titration the binding sites of the membrane for the dye, using the “end‐point” of the absorbance change or using the method of continuous variations. The number of binding sites found through these procedures is in agreement with those obtained from the binding experiments.The pH of the medium has a marked effect on the dye‐membrane interaction. Increasing the pH from 6 to 8 results in an increase of the rate of binding, in a decrease of the dissociation constant and in a decrease of the number of binding sites.The lipophilic cation, nupercaine, exerts a marked inhibition on the binding of the cationic dye and the concomitant decrease of absorbancy. The inhibition is of competitive type with aKiof 20 μM.The presence of electrolytes in the medium is required for the high affinity binding and the change of absorbance. Replacement of KCl with sucrose results in abolition of both phenomena.The results are discussed in relation to the problem of the proposed inverted polarity of the ion translocation mechanism in the submitochondrial particles. The suggestion is made that sonication of mitochondria results in a “loss of polarity” rather than in “inversion of polarity”. The kinetics of the interaction indicates a limiting step which is probably that required for the interaction of the dye with the energized site.