Brustovetsky N, Klingenberg M
Institute of Physical Biochemistry, University of Munich, Federal Republic of Germany.
J Biol Chem. 1994 Nov 4;269(44):27329-36.
In a reconstituted system, the participation of the ATP/ADP carrier (AAC) in the free fatty acid (FFA)-induced proton transport was demonstrated (i) by direct measuring of the proton transport through the membranes of AAC proteoliposomes and (ii) by monitoring of the transmembrane potential delta psi in AAC-cytochrome-c oxidase (COX)-coreconstituted proteoliposomes. FFA increased the initial rate of proton transport in AAC proteoliposomes and decreased delta psi in AAC-COX proteoliposomes. Inhibitors of AAC suppressed the effects of FFA. Without AAC or with inactive AAC, FFA cannot maintain proton leakage through the membrane. In these cases, even a small increase of delta psi was induced by FFA. These results demonstrate for the first time with purified components a participation of AAC in FFA-induced proton transport supporting an earlier suggestion (Skulachev, V.P. (1991) FEBS Lett. 294, 158-162). Mersalyl treatment of the AAC-COX proteoliposomes resulted in an increase of the AAC-mediated protonophoric action of FFA. Mersalyl also sensitized the protonophoric action of the FFA against nucleotides so that even guanine nucleotides, which are inactive in transport, become inhibitory. The effect of mersalyl is rationalized in terms of a specific interaction with cysteine 159 being attracted as anion by surrounding positive charges. This might open a gate similarly as suggested for eosin 5-maleimide interaction (Majima, E., Koike, H., Hong, Y.-M., Shinohara, Y., and Terada, H. (1993) J. Biol. Chem. 268, 22181-22187) and, thus, transform the AAC into undirectional transport mode.
在一个重组系统中，通过以下方式证明了ATP/ADP载体（AAC）参与游离脂肪酸（FFA）诱导的质子转运：（i）直接测量质子通过AAC蛋白脂质体膜的转运；（ii）监测AAC - 细胞色素c氧化酶（COX）重组蛋白脂质体中的跨膜电位Δψ。FFA增加了AAC蛋白脂质体中质子转运的初始速率，并降低了AAC - COX蛋白脂质体中的Δψ。AAC抑制剂抑制了FFA的作用。没有AAC或使用无活性的AAC时，FFA无法维持质子通过膜的泄漏。在这些情况下，FFA甚至会诱导Δψ的小幅增加。这些结果首次用纯化的组分证明了AAC参与FFA诱导的质子转运，支持了早期的一项建议（Skulachev，V.P.（1991）FEBS Lett. 294，158 - 162）。对AAC - COX蛋白脂质体进行汞撒利处理导致FFA的AAC介导的质子载体作用增强。汞撒利还使FFA对核苷酸的质子载体作用敏感，以至于即使在转运中无活性的鸟嘌呤核苷酸也变得具有抑制作用。汞撒利的作用可根据与半胱氨酸159的特异性相互作用来解释，半胱氨酸159被周围的正电荷吸引为阴离子。这可能会像对曙红5 - 马来酰亚胺相互作用所建议的那样打开一个通道（Majima，E.，Koike，H.，Hong，Y.-M.，Shinohara，Y.，和Terada，H.（1993）J. Biol. Chem. 268，22181 - 22187），从而将AAC转变为单向转运模式。
