title: Cloning, expression and characterization of theta-class glutathione S-transferase from the silkworm, Bombyx mori.
abstract: This study focused on glutathione S-transferase (GST), one of the detoxification enzymes, from the silkworm, Bombyx mori (GSTT1). A cDNA encoding a putative GST was amplified by reverse transcriptase-polymerase chain reaction and sequenced. The deduced amino acid sequence revealed 59%, 57% and 56% identities to theta-class GSTs of Musca domestica, Anopheles gambiae and Drosophila melanogaster, respectively. GSTT1 was also estimated to be close to those GSTs in a phylogenetic tree. Recombinant GST (rGSTT1) was functionally overexpressed in Escherichia coli in a soluble form, purified to homogeneity, and characterized. The pH-optimum of rGSTT1 was broad from pH 4 to 9 and rGSTT1 retained more than 75% of its original activity after incubation at pH 5-11. Incubation for 30 min at temperatures below 50 degrees C also affected the activity insignificantly. The Michaelis constant for 1-chloro-2,4-dinitrobenzene was 0.48 mM.
authors.affiliation: Laboratory of Insect Genetic Resources, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Fukuoka 812-8581, Japan. yamamok@agr.kyushu-u.ac.jp
authors.forename: Kohji
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Pingbo
authors.initials: P
authors.lastname: Zhang
authors.forename: Fumio
authors.initials: F
authors.lastname: Miake
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Kashige
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Aso
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Banno
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fujii
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; C413545:glutathione S-transferase T1; D005982:Glutathione Transferase