Busulfan-glutathione conjugation catalyzed by human liver cytosolic glutathione S-transferases.
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-alpha 1-1. Human liver microsomes and cytosol were incubated with 40 microM 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 (Vmax/K(m)) of busulfan conjugation was 0.167 microliter/min/mg with 50-1200 microM busulfan and 1 mM GSH. GSH Vmax and K(m) for busulfan conjugation were 30.6 pmol/min/mg and 312 microM, respectively. Ethacrynic acid (0.03-15 microM) inhibited cytosolic busulfan-conjugating activity with 40 microM busulfan and 1 mM GSH. Enzyme-mediated THT+ formation was decreased 97% by 15 microM ethacrynic acid with no effect on the spontaneous reaction. In incubations with affinity-purified liver GST and GST-alpha 1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 microliters/min/mg, respectively. Busulfan is a GST substrate with a high K(m) relative to concentrations achieved clinically (1-8 microM).
Gibbs
J P
JP
Department of Pharmaceutics, University of Washington, Seattle 98195, USA.
Czerwinski
M
M
Slattery
J T
JT
D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism*; D002066:Busulfan / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology
D018906:Antineoplastic Agents, Alkylating; D005982:Glutathione Transferase; D002066:Busulfan; D005978:Glutathione