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-α1-1. 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 Km for busulfan was not determined. The intrinsic clearance (Vmax/Km) of busulfan conjugation was 0.167 µl/min/mg with 50–1200 µm busulfan and 1 mm GSH. GSH Vmax and Km 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-α1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 µl/min/mg, respectively. Busulfan is a GST substrate with a high Km relative to concentrations achieved clinically (1–8 µm).
This work was supported in part by NIH Grants GM 32165, ES 07033, CA 18029, CA 47748, and HL 53750 (J. T. S.).