Cytosolic Glutathione Busulfan-Glutathione Conjugation Catalyzed by Human Liver

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-al-1. Human liver microsomes and cytosol were incubated with 40 /IM busulfan and 1 m\i (.SII. Cytosol catalyzed the formation of the GSH-husulfan tetrahydrothiophenium ion i III I ' i in a concentration-dependent man ner, 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 A„, for husulfan was not determined. The intrinsic clearance (Vnlav/Ä,„) of husulfan conjugation was 0.167 /¿1/min/mgwith 50-1200 /IM busulfan and 1 HIMGSH. GSH Vlnax and Km for busulfan conjugation were 30.6 pmol/ntin/mg and 312 /IM. respectively. Ethacrynic acid (0.03-15 /IMI inhibited cytosolic busulfan-conjugating activity with 40 /IM busulfan and 1 HIMGSH. Knzyme-mediated IIII formation was de creased 97% by 15 /IM ethacrynic acid with no effect on the spontaneous reaction. In incubations with affinity-purified liver GST and GST-al—1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 /il/min/ mg, respectively. Busulfan is a GST substrate with a high A',„ relative to concentrations achieved clinically (1-8 /IM