Tanveer A, Virji S, Andreeva L, Totty N F, Hsuan J J, Ward J M, Crompton M
Department of Biochemistry and Molecular Biology, University College London, England.
Eur J Biochem. 1996 May 15;238(1):166-72. doi: 10.1111/j.1432-1033.1996.0166q.x.
Heart and liver mitochondria contain a structure that is able to form a large non-selective pore in the inner membrane under conditions of high matrix Ca2+ and oxidant stress. The pore is blocked by cyclosporin A (CSA). In this study, rat liver mitochondria were covalently labelled with a photoactive CSA derivative in the presence and absence of the pore ligands Ca2+ and ADP. Photolabelling of a 21-kDa protein was selectively depressed by Ca2+ in a manner reversed by ADP. The protein exhibited peptidyl-prolyl cis-trans isomerase (PPIase) activity and was inhibited by CSA (Ki, 8 nM). The PPIase was associated with the outside of sonicated submitochondrial particles but dissociated in 0.5 M NaCl. When mitochondria were treated with increasing concentrations of digitonin, the 21-kDa PPIase fractionated with the matrix marker enzyme, malate dehydrogenase. A second PPIase of 18 kDa fractionated with the intermembrane-space marker, adenylate kinase. Photolabelling of the 18-kDa PPIase was unaffected by Ca2+ or ADP. The 21-kDa PPIase was digested with endoproteinase Asp-N and 11 of the peptides were N-terminally sequenced. The sequences were most similar to those of human cyclophilin-D, and it is concluded that this protein is probably the CSA receptor during pore blockade by CSA. The implications of these findings are discussed.
心脏和肝脏线粒体含有一种结构，在高基质Ca2+和氧化应激条件下，该结构能够在内膜上形成一个大的非选择性孔道。该孔道被环孢素A（CSA）阻断。在本研究中，在存在和不存在孔道配体Ca2+和ADP的情况下，用一种光活性CSA衍生物对大鼠肝脏线粒体进行共价标记。Ca2+以一种被ADP逆转的方式选择性地抑制了一种21 kDa蛋白质的光标记。该蛋白质表现出肽基脯氨酰顺反异构酶（PPIase）活性，并被CSA抑制（Ki，8 nM）。PPIase与超声处理的亚线粒体颗粒的外部相关，但在0.5 M NaCl中解离。当用浓度不断增加的洋地黄皂苷处理线粒体时，21 kDa的PPIase与基质标记酶苹果酸脱氢酶一起分级分离。另一种18 kDa的PPIase与膜间隙标记物腺苷酸激酶一起分级分离。18 kDa PPIase的光标记不受Ca2+或ADP的影响。用天冬氨酸内肽酶对21 kDa的PPIase进行消化，并对11个肽段进行N端测序。这些序列与人类亲环蛋白-D的序列最为相似，得出的结论是，该蛋白质可能是CSA在阻断孔道时的受体。讨论了这些发现的意义。
