Molecular cloning and characterization of theta-class glutathione S-transferase (GST-T) from the hermaphroditic fish Rivulus marmoratus and biochemical comparisons with alpha-class glutathione S-transferase (GST-A).
We cloned and sequenced full-length cDNA of a theta-class-like glutathione S-transferase (GST-T) from liver tissue of the self-fertilizing fish Rivulus marmoratus. The full-length cDNA of rm-GST-T was 907 bp in length containing an open reading frame of 666 bp that encoded a 221-amino acid putative protein. Its derived amino acid sequence was clustered with other vertebrate theta-class GSTs in a phylogenetic tree. The deduced amino acid sequence of theta-like rm-GST (rm-GST-T) was compared with both classes (alpha and theta) of GST and alpha-class rm-GST (rm-GST-A). Tissue-specific expression of two rm-GST mRNAs was investigated using real-time RT-PCR. To further characterize the catalytic properties of this enzyme along with rm-GST-A, we constructed the recombinant theta-like rm-GST plasmid with a 6 x His-Tag at the N-terminal of rm-GST-T cDNA. Recombinant rm-GST-T was highly expressed in transformed Escherichia coli, and its soluble fraction was purified by His-Tag affinity column chromatography. The kinetic properties and effects of pH and temperature on rm-GST-T were further studied, along with enzyme activity and inhibition effects, and compared with recombinant rm-GST-A. These results suggest that recombinant rm-GSTs such as rm-GST-A and rm-GST-T play a conserved functional role in R. marmoratus.
Lee
Young-Mi
YM
Department of Molecular and Environmental Bioscience, and the National Research Laboratory of Marine Molecular and Environmental Bioscience, Graduate School, Hanyang University, Seoul 133-791, South Korea.
Seo
Jung Soo
JS
Jung
Sang-Oun
SO
Kim
Il-Chan
IC
Lee
Jae-Seong
JS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003532:Cyprinodontiformes / Q000235:genetics* / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D057850:Hermaphroditic Organisms; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D019849:Sex Determination Processes
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D005982:Glutathione Transferase