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Reversible Conjugation of Ethacrynic Acid with Glutathione and Human Glutathione S-Transferase P1-1¹

Department of Biological Toxicology, TNO Toxicology and Nutrition Institute, P. O. Box 360, 3700 AJ Zeist, the Netherlands

² To whom requests for reprints should be addressed.

The reversibility of the conjugation reaction of the diuretic drug ethacrynic acid (EA), an ,β-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 h1 (pH 8.4).

From the fact that preincubation of GST Pl-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.

¹ This investigation was supported by the NWO-Foundation for Medical and Health Research MEDIGON (Grant 900-521-124).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/20/93. Accepted 12/16/93.

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