用荧光素磷脂酰乙醇胺直接测定线粒体内膜外表面pH(英文)

USING A FLUORESCEIN DERIVATIVE OF PHOSPHATIDYL-ETHANOLAMINE FOR DIRECT MEASUREMENT OF INTERFACIAL pH OF MITOPLAST MEMBRANES

由磷脂极性头部基因和结合水分子组成的氢键网络有利于质子沿膜表面侧向快速扩散。因而在线粒体氧化磷酸化过程中,与呼吸链电子传递相偶联的跨膜转运质子是否滞留于线粒体内膜外表面即成为一个值得探讨的课题。本文采用荧光素磷脂酰乙醇胺标记于线粒体内膜外表面,首次建立了直接测定线粒体内膜外表面pH的方法。标记后,线粒体内膜体呼吸控制率,呼吸链电子传递驱动的质子跨膜转移活性及ATP合成活性下降了近28.0％,11.6％和1.4％,表明荧光素磷指酰乙醇胺标记对线粒体氧化磷酸化活性无显著影响。尼日利亚菌素能够消除膜表面pH与介质pH之间的差异,在尼日利亚菌素存在下,利用酸滴定法得到了线粒体内膜外表面pH与荧光素磷脂酰乙醇胺荧光强度之间的关系。采用此方法测定了琥珀酸氧化引起的线粒体内膜外表面的pH降。

An original protocol for measuring outer interfacial pH of mitoplast membranes was established. FPE was inserted into mitoplast membrane by incubation in the presence of Ca2+ . The respiration-dependent proton translocation, respiratory control ratios and rates of ATP synthesis of FPE labeled mitoplasts decreased approximately 11.6 % , 28.0 % and 1.4% of those of control mitoplast respectively, suggesting that the ox -idative phosphorylation of FPE-labeled mitoplast was only slightly influenced after FPE labeling. The correlation between interface pH of mitoplast membrane and fluorescence intensity of FPE was obtained by acid titra-tion in the presence of nigericin which can collapse the pH difference between membrane interface and water bulk. The pH decrease in membrane interface of mitoplast induced by succinate oxidation could be measured by this method.