Reversible conjugation of ethacrynic acid with glutathione and human glutathione S-transferase P1-1.
Reversible conjugation of ethacrynic acid with glutathione and human glutathione S-transferase P1-1.
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发表时间：
1994-02
期刊：
影响因子：
11.2
通讯作者：
J. Ploemen;A. Schanke;B. V. Ommen;P. J. Bladeren
中科院分区：
文献类型：
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作者：
J. Ploemen;A. Schanke;B. V. Ommen;P. J. Bladeren
研究了利尿药依他尼酸（EA）（一种α，β-不饱和酮）与谷胱甘肽和谷胱甘肽S-转移酶P1-1（GST P1-1）结合反应的可逆性。当EA的谷胱甘肽缀合物与5倍摩尔过量的N-乙酰基-L-半胱氨酸或GST P1-1孵育时，观察到EA向N-乙酰基-L-半胱氨酸或GST P1-1的时间依赖性转移。随着pH值的增加，EA向N-乙酰-L-半胱氨酸转移的准一级反应速率常数从0.010 h-1（pH6.4）增加到0.040 h-1（pH7.4）和0.076 h-1（pH8.4）。从GST P1-1与1-氯-2，4-二硝基苯的预孵育将[14 C]EA的掺入从0.94 +/- 0.21（SD）减少到0.16 +/- 0.02 mol EA/mol亚基的事实，以及从胃蛋白酶消化[14 C] EA标记的酶后分离的主要放射性肽的自动Edman降解，由此推断EA与GST P1-1的半胱氨酸47发生反应。当GST P1-1被5倍摩尔过量的EA灭活时，加入过量的谷胱甘肽导致在约120 h内完全恢复催化活性。这些发现可能有几个含义。在正常的生理条件下，GST P1-1的抑制通过共价结合的EA将被逆转谷胱甘肽，留下可逆的抑制由谷胱甘肽共轭物的EA和EA本身作为主要的抑制机制;然而，当谷胱甘肽水平低的共价抑制可能是占主导地位的，导致在一个完全不同的时间过程中的抑制。
The reversibility of the conjugation reaction of the diuretic drug ethacrynic acid (EA), an alpha,beta-unsaturated ketone, with glutathione and glutathione S-transferase P1-1 (GST P1-1) has been studied. When the glutathione conjugate of EA was incubated with a 5-fold molar excess of N-acetyl-L-cysteine or GST P1-1, a time-dependent transfer of EA to N-acetyl-L-cysteine or GST P1-1 was observed. With increasing pH, the pseudo first order rate constants of transfer of EA to N-acetyl-L-cysteine increased from 0.010 h-1 (pH 6.4) to 0.040 h-1 (pH 7.4) and 0.076 h-1 (pH 8.4). From the fact that preincubation of GST P1-1 with 1-chloro-2,4-dinitrobenzene reduced the incorporation of [14C]EA from 0.94 +/- 0.21 (SD) to 0.16 +/- 0.02 mol EA/mol subunit and from automated Edman degradation of the major radioactive peptide isolated after pepsin digestion of the [14C]EA-labeled enzyme, it was concluded that the reaction of EA takes place with cysteine 47 of GST P1-1. When GST P1-1 was inactivated with a 5-fold molar excess of EA, adding an excess of glutathione resulted in full restoration of the catalytic activity in about 120 h. These findings may have several implications. Under normal physiological conditions the inhibition of GST P1-1 by covalent binding of EA would be reversed by glutathione, leaving reversible inhibition by the glutathione conjugate of EA and by EA itself as the main mechanism of inhibition; however, when glutathione levels are low the covalent inhibition might be predominant, resulting in a completely different time course for the inhibition.