Connern C P, Halestrap A P
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Biochemistry. 1996 Jun 25;35(25):8172-80. doi: 10.1021/bi9525177.
Binding of mitochondrial cyclophilin (CyP) to the inner mitochondrial membrane is induced by treatment of mitochondria with thiol reagents or oxidative stress and correlates with a sensitization to [Ca2+] of the cyclosporin A-sensitive mitochondrial permeability transition pore (MTP) [Connern, C. P., & Halestrap, A. P. (1994) Biochem. J. 303, 321-324]. Here we show that detection of the bound CyP by Western blotting is greatly enhanced by fixing the CyP to the blotting membrane with glutaraldehyde. CyP binding was only observed when mitochondria were incubated and then frozen in KSCN medium before preparation of the membrane fraction, but not when KCl medium was used. However, incubation of mitochondria (energized or deenergized) in KCl medium followed by KSCN addition immediately prior to freezing did allow CyP binding to be detected. The action of KSCN could be mimicked by guanidinium chloride, implying that the chaotropic action of these agents stabilized the bound complex. The sensitivity to [Ca2+] of the MTP in deenergized mitochondria was greatly enhanced in KSCN medium as compared to KCl medium. Binding of CyP to the mitochondrial membrane was increased by treatment with tert-butylhydroperoxide, phenylarsine oxide, and diamide and by hypoosmotic KCl medium. These conditions all increased the sensitivity of the MTP to [Ca2+]. Conditions known to increase the mitochondrial NADH/NAD+ ratio decreased CyP binding. In contrast, the effects of mitochondrial membrane potential, matrix pH, and adenine nucleotide translocase conformation on the sensitivity of the MTP to [Ca2+] were not associated with a change in CyP binding. Our data imply that there may be two independent mechanisms of altering the Ca2+ sensitivity of the MTP, one brought about by CyP binding which is stabilized by chaotropic agents and another involving additional regulatory sites on the pore complex.
线粒体亲环蛋白（CyP）与线粒体内膜的结合是通过用硫醇试剂处理线粒体或氧化应激诱导的，并且与环孢素A敏感的线粒体通透性转换孔（MTP）对[Ca2+]的敏感性增加相关[康纳恩，C.P.，& 哈利斯特拉普，A.P.（1994年）《生物化学杂志》303卷，321 - 324页]。在此我们表明，通过用戊二醛将CyP固定在印迹膜上，用蛋白质印迹法检测结合的CyP的能力大大增强。仅当线粒体在制备膜组分之前在KSCN培养基中孵育然后冷冻时才观察到CyP结合，而使用KCl培养基时则未观察到。然而，在KCl培养基中孵育线粒体（有能量或无能量），然后在冷冻前立即添加KSCN确实能检测到CyP结合。KSCN的作用可以被氯化铵模拟，这意味着这些试剂的离液序列高的作用稳定了结合复合物。与KCl培养基相比，在KSCN培养基中无能量线粒体中MTP对[Ca2+]的敏感性大大增强。用叔丁基过氧化氢、苯砷酸氧化物和二酰胺处理以及低渗KCl培养基可增加CyP与线粒体膜的结合。这些条件均增加了MTP对[Ca2+]的敏感性。已知增加线粒体NADH/NAD+比率的条件会降低CyP结合。相反，线粒体膜电位、基质pH和腺嘌呤核苷酸转位酶构象对MTP对[Ca2+]敏感性的影响与CyP结合的变化无关。我们的数据表明，可能存在两种改变MTP对Ca2+敏感性的独立机制，一种是由CyP结合引起的，其由离液序列高的试剂稳定，另一种涉及孔复合物上的其他调控位点。
