Guinea pig liver Mu-class glutathione S-transferase M1-2 cross-reacts with antibodies to both rat Mu- and theta-class glutathione S-transferases.
Two novel major heterodimeric Mu-class glutathione (GSH) S-transferases (GSTs), designated M1-2 and M1-3*, were isolated from guinea pig (gp) liver cytosol and purified to homogeneity together with a known major homodimeric Mu-class gpGSTM1-1 (reported as GST b by R. Oshino, K. Kamei, 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. Subunits of the heterodimers were inseparable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but could be completely separated by reverse-phase partition high-performance liquid chromatography. A molecular 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 to the Mu class. Western blot analysis using antisera raised against purified rat (r) GSTsA1-2 (Alpha), M1-1, P1-1 (Pi), and T2-2 (Theta) indicated that gpGSTsM1-1 and M1-3* cross-reacted only with anti-rGSTM1 antibody. However, gpGSTM1-2 cross-reacted intensely to almost the same extent with antibodies to both rGSTsM1-1 and T2-2. A homodimeric gpGSTM2-2, artificially constructed from native gpGSTM1-2 by treatment with guanidine hydrochloride followed by dialysis, intensely cross-reacted with antibodies to both the rat Mu- and Theta-class GSTs. Thus, the gpGST subunit M2 provided the first evidence for the double immuno-cross-reaction of a GST with polyclonal antibodies to two different classes of GSTs.
Hiratsuka
A
A
Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan.
Ogura
K
K
Fujioka
H
H
Sakamoto
Y
Y
Okuda
H
H
Wada
K
K
Tanaka
T
T
Nishiyama
T
T
Watabe
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D003001:Cloning, Molecular; D003429:Cross Reactions; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000302:isolation & purification; D019281:Dimerization; D005982:Glutathione Transferase / Q000138:chemical synthesis / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D006168:Guinea Pigs; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats
D000906:Antibodies; D018076:DNA, Complementary; D005982:Glutathione Transferase