Isolation, characterization, and expression in Escherichia coli of two murine Mu class glutathione S-transferase cDNAs homologous to the rat subunits 3 (Yb1) and 4 (Yb2).
The cytosolic glutathione S-transferases (GSTs, EC 2.5.1.18) are a superfamily of dimeric isoenzymes which catalyze the conjugation of electrophilic substrates with glutathione. We have isolated from a murine cell line (L929) two mu class GST cDNAs, pmGT10 (1.4 kilobases (kb] and pmGT2 (1.1 kb). Analysis of the deduced amino acid sequences revealed 93% homology between pmGT10 and the rat Yb1 (subunit 3) gene and 95% homology between pmGT2 and the rat Yb2 (subunit 4) gene. Furthermore, the 3'-untranslated regions of pmGT10 display a marked degree of homology to the 3' region of the rat Yb1 gene, while this region of pmGT2 displays marked homology to the corresponding region of the rat Yb2 gene. Using probes specific for each gene, northern blot analysis demonstrated that pmGT10 and pmGT2 hybridized to a 1.3-1.4-kb mRNA species present in L929 cells. These two murine cDNAs were subcloned into bacterial expression vectors, and enzymatically active GSTs encoded by pmGT10 (mGTmu1) or pmGT2 (mGTmu2) were purified from transformed Escherichia coli lysates. Western blot analysis of the individual GSTs produced in E. coli indicated that both genes encode GSTs which reacted with antibodies directed against mu class GST, but not with antibodies directed against alpha or pi class GSTs. The isoelectric points of these purified homodimers are 8.7 and 7.1, respectively, which are remarkably similar to those of the GST homodimers of the homologous rat subunits, 3-3 (or Yb1) and 4-4 (or Yb2), which are 8.4 and 6.9, respectively. Furthermore, the two murine subunits can form a heterodimer having a pI = 8.1 after in vitro dissociation with guanidine HCl, followed by dilution and dialysis to allow reassociation. Both homodimers of mGTmu1 and mGTmu2 and an apparent heterodimer are formed in L929 cells, as demonstrated by the isoelectric focusing profile of GST purified from this cell line. Hybridization of gene-specific probes with RNA from normal mouse tissues revealed that mGTmu1 transcripts were highly expressed in murine kidney, heart, lung, liver, and brain. Lower levels of mGTmu2 transcripts were also detected in kidney, heart, and lung. Expression of mGTmu2 RNA was not detected in murine liver or brain, which is in contrast to the regulation of the homologous rat subunit 4 (Yb2), which is expressed in liver and brain.
Townsend
A J
AJ
Medicine Branch, National Cancer Institute, Bethesda, Maryland 20892.
Goldsmith
M E
ME
Pickett
C B
CB
Cowan
K H
KH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D005796:Genes; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007739:L Cells / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D011994:Recombinant Proteins; D004247:DNA; D005982:Glutathione Transferase