Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase
Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase
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DOI：
10.1074/jbc.272.6.3346
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发表时间：
1997-02-07
影响因子：
4.8
通讯作者：
Connern, CP
中科院分区：
文献类型：
--
作者：
Halestrap, AP;Woodfield, KY;Connern, CP
氧化苯胂（PheArs）对线粒体通透性转换（MPT）的刺激比二酰胺和叔丁基过氧化氢（TBH）对线粒体失能的刺激更大，但CyP与线粒体内膜结合的增加（Concern，C.和Halestrap，A.在所测试的一系列核苷酸中，仅ADP、脱氧-ADP和ATP抑制MPT。ADP抑制涉及两个网站的K-i值约为1和25 μ M，这是独立的[Ca ~（2+）]和CyP结合。羧基糖苷（CAT）则使高亲和力位点消失。用TBH或二酰胺预处理线粒体后，ADP的Ki增加到50-100 μ M，而用PheArs或曙红马来酰亚胺预处理使Ki增加到>500 μ M;在两种情况下都只观察到一个抑制位点。已知曙红马来酰亚胺以CAT敏感的方式攻击腺嘌呤核苷酸移位酶（ANT）的Cys（159）（Majima，E.，Shinohara，Y.，Yamaguchi，N.，洪，Y。M.，和Terada，H.（1994）Biochemistry 33，9530-9536），并且在此我们证明了ANT与PheArs亲和柱的CAT敏感性结合。在腺嘌呤核苷酸耗尽的线粒体中，在存在或不存在硫醇试剂的情况下，观察到解偶联剂对MPT的刺激，这表明膜电位可能通过增加腺嘌呤核苷酸结合来抑制MPT，通过对ANT形成的影响。我们得出结论，CsA和ADP抑制孔开放在不同的方式，CsA通过取代结合CyP和ADP通过结合ANT。这两种机制的作用，以降低Ca 2+的孔的敏感性。硫醇试剂和氧化应激可以修饰ANT上的两个硫醇基团，从而通过这两种方式刺激孔开放。
Stimulation of the mitochondrial permeability transition (MPT) is 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 (Concern, 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 K-i values of about 1 and 25 mu M which were independent of [Ca2+] and CyP binding. Carboxyatractyloside (CAT) abolished the high affinity site. Following pretreatment of mitochondria with TBH or diamide, the K-i for ADP increased to 50-100 mu M, whereas pretreatment with PheArs or eosin maleimide increased the K-i to >500 mu M; only one inhibitory site was observed in both cases. Eosin maleimide is known to attack Cys(159) 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 formation. 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.