Heterologous expression of the allelic variant mu-class glutathione transferases mu and psi.
cDNA encoding the more acidic form, glutathione transferase (GST) psi, of the polymorphic Mu-class GSTs discovered in liver, was mutated in the 5'-end to create an NcoI site, facilitating cloning into the expression plasmid pKK233-2. The protein expressed from this construct has a point mutation Pro-2----Ala-2, but gives a catalytically functional protein. Back-mutation of the codon for amino acid residue 2 gave rise to a plasmid expressing the wild-type enzyme GST psi, or GST Mu1b-1b. A variant cDNA, differing only in specifying lysine rather than asparagine in position 173 of the coding region, was generated by site-directed mutagenesis. The variant sequence corresponds to another cDNA clone isolated from a human liver cDNA library and expresses the near-neutral GST mu, or GST Mu1a-1a. The two recombinant proteins GST Mu1a-1a and GST Mu1b-1b, by physicochemical as well as kinetic criteria, were found to be indistinguishable from GST mu and GST psi respectively, isolated from human liver. It is therefore concluded that the recombinant proteins correspond to the allelic variants observed in the human population. The two forms have different isoelectric points and correspond to the allelic variants observed in the human population. The two forms have different isoelectric points and their protein subunits can be separated by h.p.l.c. on a reverse-phase column. With standard substrates and inhibitors no differences in kinetic parameters between the two variants were detected. The mutated GST Mu1b-1b (Pro-2----Ala) was not significantly different in catalytic properties from the wild-type enzyme, even though Pro-2 is a well conserved amino acid residue in the known Mu-class GSTs.
Widersten
M
M
Department of Biochemistry, University of Uppsala, Biomedical Center, Sweden.
Pearson
W R
WR
Engström
A
A
Mannervik
B
B
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D014644:Genetic Variation; D005982:Glutathione Transferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D007527:Isoenzymes; D011994:Recombinant Proteins; D005982:Glutathione Transferase