Cloning, expression, and characterization of a class-mu glutathione transferase from human muscle, the product of the GST4 locus.
A class-mu glutathione transferase cDNA clone, GTHMUS, was isolated from human myoblasts and its sequence was determined. The sequence predicts a protein of molecular weight 25,599 whose 24 amino-terminal residues are identical to those of the class-mu isoenzyme expressed from the GST4 locus. The GTHMUS cDNA shares 93.7% nucleotide sequence identity with a human liver cDNA clone, GTH411, that is encoded at the GST1 locus. Comparison of the liver and muscle cDNA sequences shows two regions of remarkable sequence conservation: a 140-nucleotide region in the 5' coding portion of the molecule that has a single silent nucleotide substitution, and a 550-nucleotide region, including the entire 3' noncoding region, that has only three nucleotide substitutions or deletions. This sequence conservation suggests that gene conversion has occurred between the human GST1 and GST4 glutathione transferase gene loci. The human muscle and liver glutathione transferase clones GTHMUS and GTH411 have been expressed in Escherichia coli. The kinetic mechanism of the muscle enzyme was examined in product inhibition studies. The inhibition patterns are best modeled by a steady-state ordered bi-bi reaction mechanism. Glutathione is the first substrate bound and chloride ion is the first product released. Chloride ion inhibits the muscle enzyme.
Vorachek
W R
WR
Department of Biochemistry, University of Virginia, Charlottesville 22908.
Pearson
W R
WR
Rule
G S
GS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015342:DNA Probes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D015342:DNA Probes; D007527:Isoenzymes; D004247:DNA; D005982:Glutathione Transferase; D005978:Glutathione