Eriksson O, Fontaine E, Bernardi P
Consiglio Nazionale delle Ricerche Unit for the Study of Biomembranes and the Laboratory of Biophysics and Membrane Biology, Department of Biomedical Sciences, University of Padova Medical School, Viale Giuseppe Colombo 3, I-35121 Padova, Italy.
J Biol Chem. 1998 May 15;273(20):12669-74. doi: 10.1074/jbc.273.20.12669.
We have investigated the role of arginine residues in the regulation of the mitochondrial permeability transition pore, a cyclosporin A-sensitive inner membrane channel. Isolated rat liver mitochondria were treated with the arginine-specific chemical reagent 2, 3-butanedione or phenylglyoxal, followed by removal of excess free reagent. After this treatment, mitochondria accumulated Ca2+ normally, but did not undergo permeability transition following depolarization, a condition that normally triggers opening of the permeability transition pore. Inhibition by 2,3-butanedione and phenylglyoxal correlated with matrix pH, suggesting that the relevant arginine(s) are exposed to the matrix aqueous phase. Inhibition by 2,3-butanedione was potentiated by borate and was reversed upon its removal, whereas inhibition by phenylglyoxal was irreversible. Treatment with 2,3-butanedione or phenylglyoxal after induction of the permeability transition by Ca2+ overload resulted in pore closure despite the presence of 0.5 mM Ca2+. At concentrations that were fully effective at inhibiting the permeability transition, these arginine reagents (i) had no effect on the isomerase activity of cyclophilin D and (ii) did not affect the rate of ATP translocation and hydrolysis, as measured by the production of a membrane potential upon ATP addition in the presence of rotenone. We conclude that reaction with 2,3-butanedione and phenylglyoxal results in a stable chemical modification of critical arginine residue(s) located on the matrix side of the inner membrane, which, in turn, strongly favors a closed state of the pore.
我们研究了精氨酸残基在调节线粒体通透性转换孔（一种对环孢素A敏感的内膜通道）中的作用。用精氨酸特异性化学试剂2,3 - 丁二酮或苯乙二醛处理分离的大鼠肝脏线粒体，随后去除过量的游离试剂。经过这种处理后，线粒体正常积累Ca2 +，但在去极化后不会发生通透性转换，而去极化通常会触发通透性转换孔的开放。2,3 - 丁二酮和苯乙二醛的抑制作用与基质pH相关，这表明相关的精氨酸暴露于基质水相中。2,3 - 丁二酮的抑制作用被硼酸盐增强，去除硼酸盐后抑制作用逆转，而苯乙二醛的抑制作用是不可逆的。在Ca2 +过载诱导通透性转换后用2,3 - 丁二酮或苯乙二醛处理，尽管存在0.5 mM Ca2 +，仍导致孔关闭。在完全有效抑制通透性转换的浓度下，这些精氨酸试剂（i）对亲环蛋白D的异构酶活性没有影响，并且（ii）不影响ATP转运和水解的速率，这是通过在存在鱼藤酮的情况下添加ATP时产生的膜电位来测量的。我们得出结论，与2,3 - 丁二酮和苯乙二醛的反应导致位于内膜基质侧的关键精氨酸残基发生稳定的化学修饰，这反过来又强烈有利于孔的关闭状态。
