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Busulfan-Glutathione Conjugation Catalyzed by Human Liver Cytosolic Glutathione S-Transferases¹

Department of Pharmaceutics [J. P. G., M. C., J. T. S.], University of Washington, Seattle, Washington 98195, and the Fred Hutchinson Cancer Research Center [J. T. S.], Seattle, Washington 98104

We have examined the catalytic activity of glutathione S-transferases (GST) in the conjugation of busulfan with glutathione (GSH) in human liver cytosol, purified human liver GST, and cDNA-expressed GST-1-1. Human liver microsomes and cytosol were incubated with 40 µM busulfan and 1 mM GSH. Cytosol catalyzed the formation of the GSH-busulfan tetrahydrothiophenium ion (THT⁺) in a concentration-dependent manner, whereas microsomes lacked activity. The total and spontaneous rates of THT⁺ formation increased with pH (pH range, 6.50–7.75), with the maximum difference at pH 7.4. Due to the limited aqueous solubility of busulfan, a K_m for busulfan was not determined. The intrinsic clearance (V_max/K_m) of busulfan conjugation was 0.167 µl/min/mg with 50–1200 µM busulfan and 1 mM GSH. GSH V_max and K_m for busulfan conjugation were 30.6 pmol/min/mg and 312 µM, respectively. Ethacrynic acid (0.03–15 µM) inhibited cytosolic busulfan-conjugating activity with 40 µM busulfan and 1 mM GSH. Enzyme-mediated THT⁺ formation was decreased 97% by 15 µM ethacrynic acid with no effect on the spontaneous reaction. In incubations with affinity-purified liver GST and GST-1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 µl/min/mg, respectively. Busulfan is a GST substrate with a high K_m relative to concentrations achieved clinically (1–8 µM).

¹ This work was supported in part by NIH Grants GM 32165, ES 07033, CA 18029, CA 47748, and HL 53750 (J. T. S.).

² To whom requests for reprints should be addressed, at Department of Pharmaceutics, Box 357610, University of Washington, Seattle, WA 98185-3576. Phone: (206) 543-7736; Fax: (206) 543-3204; E-mail: jts@u.washington.edu.

Received 2/12/96. Accepted 6/18/96.

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