Rat Liver Glutathione &Transferases NUCLEOTIDE SEQUENCE ANALYSIS OF A Ybl cDNA CLONE AND PREDICTION OF THE COMPLETE AMINO ACID SEQUENCE OF THE Ybl SUBUNIT*

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb, mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Ybl subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with M, = 25,919. The NHz-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transfer- ase A, a Yb, homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) 2. BioZ. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5’- untranslated

The rat liver glutathione S-transferases represent a family of isozymes which catalyze the conjugation of glutathione to various electrophilic ligands. These proteins also, bind with high affinity various exogenous hydrophobic compounds as well as potentially toxic compounds such as bilirubin and heme (1)(2)(3). At least 10 cytosolic rat liver glutathione stransferases have been purified and characterized to various extents (3)(4)(5)(6). All isozymes appear to be heterodimers or homodimers comprised of subunits designated Ya, Ya, Yb, Yc, and Yn which can be distinguished electrophoretically on one-dimensional sodium dodecyl sulfate-polyacrylamide gels Although the nucleotide sequences of cDNA clones complementary to the Ya and Yc mRNAs have been determined (7lo), there is no sequence data to date on a cDNA clone complementary to the rat liver glutathione S-transferase Yb * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (4)(5)(6).
1[ To whom correspondence may be addressed. mRNA family. Similarly, amino acid sequence data of purified Yb subunits are also very limited. Only recently have the first 19 NHz-terminal amino acids of the Yb, and Y b z subunits of the rat liver glutathione S-transferases been reported (11). Although the Yb subunits have similar electrophoretic mobilities and are immunologically related, the chemical composition of these subunits has not been established. The construction of full length cDNA clones complementary to specific Yb mRNAs is crucial toward determining the primary structure of the Yb subunits and elucidating the mechanisms by which the Yb gene family is regulated by xenobiotics.
In earlier work from our laboratory, we described the construction and characterization of a truncated cDNA clone, pGTA/C36, which is complementary to a Yb mRNA (7). In the present study, we have utilized this clone to screen a cDNA library constructed from purified glutathione S-transferase mRNAs. We have identified a recombinant clone, pGTA/C44, which contains a nearly full length cDNA insert complementary to the Ybl mRNA. The entire nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the corresponding Ybl subunit has been deduced. Analysis of the nucleotide sequence of the Ybl cDNA clone indicates it shares significant sequence homology with a cDNA clone, pGT55, which is complementary to mouse liver glutathione S-transferase mRNA (12). However, no significant sequence homology was found between the rat liver Ybl clone and the Ya or Yc cDNA clones described previously by our laboratory (7,10). These latter data suggest that the rat liver glutathione S-transferase Ya-Yc subunits and the Yb subunits are derived from different gene families.

MATERIALS AND METHODS AND RESULTS~
DNA Sequence Analysis of pGTAIC44 and the. Deduced Amino Acid Sequence of the Ybl Subunit-Sequence analysis of pGTA/C44 was carried out using the chemical sequencing procedure of Maxarn and Gilbert (19). The entire nucleotide sequence of pGTA/C44 is illustrated in Fig. 1 along with the deduced amino acid sequence of the Yb subunit. The length of the cDNA insert is 1038 base pairs minus the dC tails. The subjected to sequence analysis using the Maxam-Gilbert chemical sequencing procedure (19). Restriction endonuclease sites (see Fig. 1 in Miniprint) used for 5'-end labeling were the N e d , BamHI, BglII, and StuI sites. The DNA sequence was determined in the 5' and 3' directions from these sites. Restriction endonuclease sites used for 3'-end labeling were the SphI and PstI sites. For the SphI site sequence, analysis proceeded in both the 5' and 3' directions. For the PstI sites, both PstI fragments (748 and 290 base pairs) were isolated, 3'-end-labeled, subjected to secondary restriction endonuclease digestion, and sequenced from the end label. The sequence of all fragments was determined at least twice, and approximately 80% of the sequence has been determined in both directions. Recently, Frey et al. (11) have determined the NHp-terminal sequences of glutathione S-transferases A, C, and X. The NHz-terminal sequences of transferases A and X are presented in Table I (see Miniprint) along with the deduced amino acid sequence obtained from DNA sequence analysis of pGTA/C44. In the first 19 amino acids, there exist five amino acid differences between transferase A and transferase X (Yb, homodimer). These differences occur at positions 3 (Ile-Thr), 9 (Val-Ile), 13 (Thr-Ala), 15 (Pro-Ala), and 19 (Leu-Phe). At every divergent position, the amino acid sequence deduced from pGTA/C44 agrees with the NHz-terminal sequence determined for glutathione S-transferase A. These data indicate that the cDNA insert in pGTA/C44 is complementary to the Ybl mRNA rather than the Ybz mRNA.
Comparison of Sequence Homology of the Rat Liver Glutathione S-Transferase Ybl Subunit with Mouse Liver Glutathione S-Transferases-Pearson et al. (12) have constructed and characterized a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase. One hundred ninety-four bp near the 5'-end of the mRNA sequence were determined and are presented in Fig. 2 along with the nucleotide sequence of the corresponding region in pGTA/C44. As can be seen from this figure, there is an 85% nucleotide sequence homology between the mouse and rat sequence in this region. The NHz-terminal amino acid sequences of the mouse liver glutathione S-transferase 8.7 and 9.3 isozymes are presented in Fig. 3. The NHz-terminal sequence of glutathione S-transferase 8.7 was determined by protein sequencing techniques, whereas the NHz-terminal amino acid sequence of glutathione S-transferase 9.3 was deduced from the nucleotide sequence of pGT55 as well as from conventional protein sequencing techniques (12). There is an 86% amino acid sequence homology between the rat liver Ybl subunit and the mouse liver glutathione S-transferase 9.3 subunit over the first 72 NHz-terminal amino acids, whereas the Ybl subunit shares an 80% sequence homology with glutathione S-transferase 8.7 over the first 40 NH2-terminal amino acids.

DISCUSSION
In this investigation, we have identified a cDNA clone, pGTA/C44, which is complementary to a mRNA specific for a Yb subunit of the rat liver glutathione S-transferases. The DNA sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb, subunit has been deduced. The NH,-terminal amino acid sequence deduced from the DNA sequence of pGTA/C44 agrees with the NH,terminal amino acid sequence of the Ybl subunit determined by Frey et al. (11) using conventional protein sequencing techniques. These data provide the first detailed sequence analysis of a cDNA clone complementary to the rat liver glutathione S-transferase Yb mRNA family.
Although the rat liver glutathione S-transferase Ya and Yc cDNA clones have a 66% nucleotide sequence homology (9, lo), the Ybl mRNA appears to have little sequence homology with the Ya or Yc mRNAs. Similarly, there is little amino acid sequence homology between the Ybl subunit and the Ya or Yc subunits. The lack of significant homology between the Ya-Yc mRNAs and the Ybl mRNA suggests these mRNAs are transcriptional products of distinct gene families which have evolved independently. These data are consistent with previous work from our laboratory which has demonstrated that the Ya and Yb mRNAs are regulated independently by 3-methylcholanthrene and phenobarbital (7).
The lack of significant amino acid sequence homology is not surprising given that the immunochemical and catalytic properties of the Ya, Yc, and Yb subunits are quite distinct. For example, polyclonal antibodies raised against the Yb subunit family do not cross-react with the Ya or Yc subunits. Similarly, the glutathione S-transferases comprised of Ybl and Yb2 subunits have high activity toward bromosulfophthalein and trans-4-phenyl-3-butene-2-one, respectively, whereas the Ya and Yc subunits have high steroid isomerase  FIG. 2 (left). DNA sequence comparison of pGTA/C44 with mouse liver pGT55. The DNA sequence of mouse liver pGT55 (12) is compared to the corresponding region in pGTA/C44 (nucleotides 71-257). Over this region of both sequences, there is an 85% nucleotide sequence homology.
FIG. 3 (right). Protein sequence homology between the rat liver Ybl subunit and the mouse liver glutathione S-transferases 8.7 and 9.3. The NH2-terminal protein sequence of the mouse liver glutathione Stransferase 9.3 has been determined by DNA sequence analysis of pGT55 and protein sequencing techniques (12), whereas the NHB-terminal sequence of the mouse liver glutathione S-transferase 8.7 has been determined entirely by protein sequencing techniques (12). There is an 86% sequence homology between the rat liver Ybl subunit and mouse liver glutathione S-transferase 9.3 over the first 72 amino acids and an 80% sequence homology between the rat liver Yb, subunit and the mouse liver glutathione S-transferase 8.7. and cumene hydroperoxidase activity, respectively (20, 21).
Recently, Pearson et al. (12) reported the isolation of a cDNA clone, pGT55, which i s complementary to mouse liver glutathione S-transferase 9.3 mRNA but also shares significant sequence homology with the glutathione S-transferase 8.7 isozyme. Interestingly, like glutathione S-transferase A (Ybl homodimer), the mouse liver glutathione S-transferase 8.7 has high activity toward 1,2-dichloro-4-dinitrobenzene whereas glutathione S-transferase 9.3 is approximately 4-fold less active with this substrate. These data indicate that mouse glutathione S-transferase 8.7 has catalytic properties similar to the rat glutathione S-transferase Yb, subunit, whereas mouse glutathione S-transferase 9.3 may be more similar to the rat Y b , subunit. Finally, these data suggest that mouse liver glutathione S-transferases (8.7 and 9.3) and the rat liver glutathione S-transferase Yb subunits have evolved from a common ancestral gene.
The construction and characterization of a full length Ybl cDNA clone will facilitate the isolation and characterization of the structural genes encoding this family of glutathione Stransferases as well as elucidate the mechanisms by which the genes are regulated by various xenobiotics.