GUINEA-PIG LIVER MU-CLASS GLUTATHIONE-S-TRANSFERASE M1-2 CROSS-REACTSWITH ANTIBODIES TO BOTH RAT MU-CLASS AND THETA-CLASS GLUTATHIONE S-TRANSFERASES

Two novel major heterodimeric Mu-class glutathione (GSH) S-transferase s (GSTs), designated M1-2 and M1-3, were isolated from guinea pig (gp ) Fiver cytosol and purified to homogeneity together with a known majo r homodimeric Mu-class gpGSTM1-1 (reported as GST b by R. Oshino, K. K amei, M. Nishioka, and M. Shin, 1990, J. Biochem. 107, 105-110). These three gpGSTs were quantitatively retained on an S-hexyl-GSH affinity column and separated as homogeneous proteins by chromatofocusing. Subu nits of the heterodimers were inseparable on sodium dodecyl sulfate-po lyacrylamide gel electrophoresis, but could be completely separated by reverse-phase partition high-performance liquid chromatography. A mol ecular cloning study demonstrated that the gpGST subunit M2 consisted of 217 amino acid residues with a calculated molecular mass of 25,562 and shared 84% identity in overall amino acid sequence with gpGSTM1-1. N-terminal amino acid sequences of peptides from the gpGST subunit M3 with a blocked N-terminus strongly suggested that it should belong t o the Mu class. Western blot analysis using antisera raised against pu rified rat (r) GSTsA1-2 (Alpha), M1-1, P1-1 (Pi), and T2-2 (Theta) ind icated that gpGSTsM1-1 and M1-3 cross-reacted only with anti-rGSTM1 a ntibody. However, gpGSTM1-2 cross-reacted intensely to almost the same extent with antibodies to both rGSTsM1-1 and T2-2, A homodimeric gpGS TM2-2, artificially constructed from native gpGSTM2-2 by treatment wit h guanidine hydrochloride followed by dialysis, intensely cross-reacte d with antibodies to both the rat Mu-and Theta-class GSTs. Thus, the g pGST subunit M2 provided the first evidence for the double immuno-cros s-reaction of a GST with polyclonal antibodies to two different classe s of GSTs. (C) 1998 Academic Press.