Halestrap A P, Woodfield K Y, Connern C P
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
J Biol Chem. 1997 Feb 7;272(6):3346-54. doi: 10.1074/jbc.272.6.3346.
Stimulation of the mitochondrial permeability transition (MPT) in de-energized mitochondria by phenylarsine oxide (PheArs) is greater than that by diamide and t-butylhydroperoxide (TBH), yet the increase in CyP binding to the inner mitochondrial membrane (Connern, C. P. and Halestrap, A. P. (1994) Biochem. J. 302, 321-324) is less. From a range of nucleotides tested only ADP, deoxy-ADP, and ATP inhibited the MPT. ADP inhibition involved two sites with Ki values of about 1 and 25 microM which were independent of [Ca2+] and CyP binding. Carboxyatractyloside (CAT) abolished the high affinity site. Following pretreatment of mitochondria with TBH or diamide, the Ki for ADP increased to 50-100 microM, whereas pretreatment with PheArs or eosin maleimide increased the Ki to >500 microM; only one inhibitory site was observed in both cases. Eosin maleimide is known to attack Cys159 of the adenine nucleotide translocase (ANT) in a CAT-sensitive manner (Majima, E., Shinohara, Y., Yamaguchi, N., Hong, Y. M., and Terada, H. (1994) Biochemistry 33, 9530-9536), and here we demonstrate CAT-sensitive binding of the ANT to a PheArs affinity column. In adenine nucleotide-depleted mitochondria, no stimulation of the MPT by uncoupler was observed in the presence or absence of thiol reagents, suggesting that membrane potential may inhibit the MPT by increasing adenine nucleotide binding through an effect on the ANT conformation. We conclude that CsA and ADP inhibit pore opening in distinct ways, CsA by displacing bound CyP and ADP by binding to the ANT. Both mechanisms act to decrease the Ca2+ sensitivity of the pore. Thiol reagents and oxidative stress may modify two thiol groups on the ANT and thus stimulate pore opening by both means.
苯胂氧化物（PheArs）对去能线粒体中线粒体通透性转换（MPT）的刺激作用大于二酰胺和叔丁基过氧化氢（TBH），然而环孢菌素（CyP）与线粒体内膜结合的增加量较少（康纳恩，C.P.和哈利斯特雷普，A.P.（1994年）《生物化学杂志》302卷，321 - 324页）。在所测试的一系列核苷酸中，只有ADP、脱氧ADP和ATP能抑制MPT。ADP的抑制作用涉及两个位点，其解离常数（Ki）值约为1和25微摩尔，这两个位点与[Ca2 +]和CyP结合无关。羧基苍术苷（CAT）消除了高亲和力位点。用TBH或二酰胺预处理线粒体后，ADP的Ki增加到50 - 100微摩尔，而用PheArs或曙红马来酰亚胺预处理则使Ki增加到>500微摩尔；在这两种情况下均只观察到一个抑制位点。已知曙红马来酰亚胺以对CAT敏感的方式攻击腺嘌呤核苷酸转位酶（ANT）的半胱氨酸159位点（真岛，E.，筱原，Y.，山口，N.，洪，Y.M.和寺田，H.（1994年）《生物化学》33卷，9530 - 9536页），在此我们证明ANT与PheArs亲和柱的结合对CAT敏感。在腺嘌呤核苷酸耗尽的线粒体中，无论有无硫醇试剂，解偶联剂均未观察到对MPT的刺激作用，这表明膜电位可能通过影响ANT构象增加腺嘌呤核苷酸结合来抑制MPT。我们得出结论，环孢菌素A（CsA）和ADP以不同方式抑制孔开放，CsA通过取代结合的CyP，而ADP通过与ANT结合。两种机制均起到降低孔对Ca2 +的敏感性的作用。硫醇试剂和氧化应激可能修饰ANT上的两个硫醇基团，从而通过两种方式刺激孔开放。
