Leakage of membrane vesicle contents: Determination of mechanism using fluorescence requenching
Leakage of membrane vesicle contents: Determination of mechanism using fluorescence requenching
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DOI：
10.1016/s0006-3495(95)80066-4
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发表时间：
1995-11-01
影响因子：
3.4
通讯作者：
White, SH
中科院分区：
文献类型：
--
作者：
Ladokhin, AS;Wimley, WC;White, SH
抗微生物肽和毒素等试剂可使膜囊泡透化，从而以分级或全或无的方式引起截留内容物的泄漏。确定哪种渗漏模式是诱导的是理解膜透化的分子机制的重要步骤。Wimley等人（1994，Protein Sci. 3：1362-1378）已经开发了一种用于区分两种模式的荧光方法，该方法利用染料/猝灭剂对8-氨基萘-1，3，6三磺酸（ANTS）/对二甲苯-双-溴化吡啶（DPX），而通常不需要从释放的内容物中物理分离囊泡。他们的“重新激发"方法通过荧光变化建立了释放模式，当DPX被外部加入到已经释放了部分内容物的囊泡溶液中时，荧光变化发生。然而，如最初所述的再猝灭方法忽略了优先释放染料或猝灭剂的可能性。在此，我们扩展了该方法的理论，考虑了优先释放和分级泄漏的影响，利用该理论可以估算阳离子猝灭剂DPX和阴离子染料8-氨基萘-1，3，6-三磺酸的释放速率之比。对于分级泄漏，我们表明标记物的释放与标准泄漏测定中观察到的荧光变化不一致。对于自猝灭染料也是如此，这意味着1）释放的材料量将被高估，2）动力学将是非指数的，并具有人为的高表观速率。我们展示了如何扩展的重调分析允许泄漏实验的结果进行校正的文物，从分级和优先泄漏。实验证据的存在肽诱导的优先梯度泄漏DPX从中性和阴离子囊泡。
Agents such as antimicrobial peptides and toxins can permeabilize membrane vesicles to cause leakage of entrapped contents in either a graded or an all-or-none fashion. Determination of which mode of leakage is induced is an important step in understanding the molecular mechanism of membrane permeabilization. Wimley et al. (1994, Protein Sci. 3:1362-1378) have developed a fluorescence method for distinguishing the two modes that makes use of the dye/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) without the usual need for the physical separation of vesicles from released contents. Their ''requenching'' method establishes the mode of release through the fluorescence changes that occur when DPX is added externally to a solution of vesicles that have released some fraction of their contents. However, the requenching method as originally stated ignored the possibility of preferential release of dye or quencher. Here we extend the theory of the method to take into account preferential release and the effects of graded leakage, The ratio of the rates of release of the cationic quencher DPX and anionic dye 8-aminonapthalene-1,3,6 trisulfonic acid can be estimated by means of the theory. For graded leakage, we show that the release of the markers does not coincide with the fluorescence changes observed in the standard leakage assay. This is true for self-quenching dyes as well and means that 1) the amount of released material will be overestimated and 2) the kinetics will be nonexponential and have artificially high apparent rates. We show how the extended requenching analysis allows the results of leakage experiments to be corrected for artifacts that result from graded and preferential leakage. Experimental evidence is presented for the existence of peptide-induced preferential graded leakage of DPX from both neutral and anionic vesicles.