title: Purification, biochemical characterization, and cDNA cloning of a glutathione S-transferase from the red imported fire ant, Solenopsis invicta.
abstract: A glutathione S-transferase (GST) was purified 266-fold from adult workers of the red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae) by affinity chromatography and preparative isoelectric focusing. The purified enzyme appeared as a single band on SDS-PAGE and had a Mr of 25.5 kDa. Steady state kinetics assays of the enzyme with 1-chloro-2,4-dinitrobenzene as substrate were conducted. The Vmax, Km CDNB, Km GSH, kcat, kcat/Km CDNB, and kcat/Km GSH for the purified fire ant GST were 87.4 micromol/min/mg, 0.13 mM, 0.84 mM, 74.5 s(-1), 573.1 mM(-1) s(-1), and 88.7 mM(-1) s(-1), respectively. An internal fragment of the enzyme, released by endoproteinase Lys-C digestion, was sequenced and used to design a degenerate oligonucleotide primer. Purified cDNA from a lambda-phage expression library produced from worker fire ants was used as template to amplify a fragment of the GST transcript which was subsequently cloned and sequenced. 5' and 3' rapid amplification of cDNA ends were subsequently conducted after cDNA production by RT-PCR of mRNA from adult worker fire ants. An open reading frame comprising 202 amino acids with a calculated molecular weight of 23,433 and a theoretical pI of 7.84 was located within the transcript beginning at nucleotide 85 and terminating in a stop codon at nucleotide 691. The transcript contained 5' and 3' untranslated regions of 84 and 296 nucleotides, respectively. The sequences of the internal fragments from the purified fire ant GST were identical to corresponding translated regions of the transcript (nucleotides 214-258, and 457-477). Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme was most closely related to sigma class GSTs.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001000:Ants / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D004137:Dinitrochlorobenzene; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D005978:Glutathione