BUSULFAN CONJUGATION BY GLUTATHIONE S-TRANSFERASE-ALPHA, S-TRANSFERASE-MU, AND S-TRANSFERASE-PI

Busulfan is eliminated by glutathione S-transferase (GST)-catalyzed co njugation with glutathione (GSH), We have characterized the busulfan-c onjugating activity of purified human liver GSTA1-1, GSTA1-2, GSTA2-2, GSTM1-1, and placental GSTP1-1, Isoforms were purified from cytosol b y GSH-affinity chromatography and chromatofocusing. In addition, the b usulfan-conjugating activity of cDNA-expressed GTH1 and GTH2, correspo nding to GSTA1-1 and GSTA2-2, were characterized, The major product of busulfan conjugation, a thiophenium ion (THT+), was assayed by GC/MS after conversion to tetrahydrothiophene (THT). THT+ formation rate inc reased linearly with busulfan concentration up to its solubility limit for all GST isoforms. Because V-max and K-M could not be determined s eparately, the slope of the velocity vs. substrate concentration plot, V-max/K-M, was used to compare isoform activities. V-max/K-M for GSTA 1-1 was 7.95 mu l/min/mg protein, the highest busulfan-conjugating act ivity of all human liver and placenta isoforms evaluated, GSTM1-1 and GSTP1-1, respectively, had 46% and 18% of the activity of GSTA1-1. Sin ce the polymorphic mu-class GST catalyzed busulfan conjugation, we exa mined busulfan clearance in 50 patients undergoing high-dose busulfan before bone marrow transplantation. Busulfan clearance was normally di stributed, suggesting that GSTM1-1 does not contribute significantly t o the elimination of busulfan from the body. We conclude that GSTA1-1 is the major isoform catalyzing busulfan conjugation, whereas GSTM1-1 and GSTP1-1 may be important in the protection of specific cells.