Cloning and expression of a cDNA for mu-class glutathione S-transferase from rabbit liver.
A mu-class glutathione S-transferase (GST) cDNA clone, pHMB1, from rabbit liver has been constructed, using a 748-base-pair fragment of GST Yb1 cDNA as a probe. The nucleotide sequence of pHMB1 has been determined, and the complete amino acid sequence has been deduced. Recombinant clone pHMB1 contains a cDNA insert of 1443 base pairs with 654 nucleotides of open reading frame, 33 nucleotides of 5'-untranslated region, and 756 nucleotides of 3'-untranslated region. The open reading frame encodes a polypeptide (rbGST mu I) comprising 218 amino acids with molecular weight of 25,417. Compared to published mu-class GST sequences, rbGST mu I is 73 and 77% identical to rat Yb1 and human GST4 in amino acid sequence, respectively. The pHMB1 was expressed in Escherichia coli using expression vector pIH821 and the expressed GST was purified as a single band on polyacrylamide gel electrophoresis by maltose- and glutathione-affinity column chromatography. Rabbit liver GST protein expressed by this system was catalytically active. The functional characterization was done on the expressed protein. The rabbit liver GST expressed in E. coli showed greater activity toward 1,2-dichloro-4-nitrobenzene than mu-class isozymes in rabbit hepatic tissue (T. Primiano and R.F. Novak (1993) Arch. Biochem. Biophys. 301, 404-410). Enzymatic activity of expressed protein toward the substrate 1-chloro-2,4-dinitrobenzene was inhibited by triethyltin bromide, Cibacron blue, triphenyltin chloride, bromosulfophthalein, and hematin. RNA blot hybridization demonstrated that the pHMB1 mRNA was well expressed in rabbit liver, brain, and kidney.
Lee
S H
SH
Department of Biochemistry, College of Medicine, Hanyang University, Seoul, Korea.
Lee
S H
SH
Han
J S
JS
Kim
Y S
YS
Koh
J K
JK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010957:Plasmids; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D005982:Glutathione Transferase