CLONING, EXPRESSION, AND BIOCHEMICAL-CHARACTERIZATION OF A FUNCTIONALLY NOVEL ALPHA-CLASS GLUTATHIONE-S-TRANSFERASE WITH EXCEPTIONAL ACTIVITY IN THE GLUTATHIONE CONJUGATION OF ROXY-9,10-OXY-7,8,9,10-TETRAHYDROBENZO(A)PYRENE())

The present study describes cDNA cloning, expression, and kinetic char acterization of the two subunits of a murine alpha-class glutathione ( GSH) S-transferase (GST) isoenzyme (previously designated as GST 9.5), which, unlike other alpha-class mammalian GSTs, is exceptionally effi cient in the GSH conjugation of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9, 10 (a)pyrene [(+)-anti-BPDE] [X. Hu, S. K. Srivastava, H. Xia, Y. C. A wasthi, and S. V. Singh (1996) J. Biol. Chem. 271, 32684-32688]. The c DNAs for both subunits of GST 9.5 (GST 9.5-1 and GST 9.5-2) were clone d by RT-PCR. The deduced amino acid sequences of GST 9.5-1 and GST 9.5 -2 clones were identical to those of mGSTA1 and mGSTA2, respectively. Both these subunits were expressed in Escherichia coli to determine th e relationships between recombinant mGSTA1-1 and mGSTA2-2 and correspo nding subunits of tissue-isolated GST 9.5. The pi values of recombinan t mGSTA1-1 and mGSTA2-2 (9.49 and 9.45, respectively) were similar to that of the tissue-isolated isoenzyme (pl 9.5). The reverse-phase HPLC elution profiles and immunological cross-reactivities of recombinant mGSTA1-1 and mGSTA2-2 were also similar to those of the corresponding subunits of tissue-isolated GST 9.5. The catalytic efficiency of recom binant mGSTA1-1 toward (+)-anti-BPDE, 131 mM(-1) s(-1), was approximat ely 9.5- to 655-fold higher compared with tissue-isolated mGSTP1-1, mG STA3-3, mGSTM1-1, and mGSTA4-4. Moreover, the catalytic efficiency of mGSTA1-1 toward (+)-anti-BPDE was about 3.3-fold higher compared with recombinant mGSTA2-2. The mGSTA1 and/or mGSTA2 subunits were expressed to varying degrees in female A/J mouse tissues. For example, mGSTA1, but not mGSTA2, subunit expression was observed in the skin, which is a target organ for benzo(a)pyrene (BP)-induced cancer in mice. On the other hand, the expression of either mGSTA1 or mGSTA2 subunit could no t be detected in the lung, which is another target organ for BP-induce d cancer in mice. Interestingly, relatively large amounts of both mGST A1 and mGSTA2 subunits were detected in the kidney. In conclusion, the results of the present study clearly indicate that the Al-type subuni t of GST 9.5 is responsible for its exceptional catalytic efficiency i n the GSH conjugation of (+)-anti-BPDE, which is the ultimate carcinog en of widespread environmental pollutant BP. (C) 1998 Academic Press.