Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/5/2003
Publication Date: 10/21/2003
Citation: Valles, S.M., Perera, O. P., Strong, C. A. 2003. Purification, biochemical characterization, and cDNA cloning of a glutathione S-transferase from the red imported fire ant, Solenopsis invicta. Insect Biochemistry and Molecular Biology. v. 33. p. 981-988. Interpretive Summary: The red imported fire ant was introduced into the United States in the 1930s and currently infests about 300 million acres. It causes significant economic losses in livestock and agricultural production and poses a serious threat to human health. Annually, 50% of people in infested areas are stung, occasionally with fatal consequences. Although insecticides have been used to control fire ants for decades, very little research has been conducted on their detoxification systems. Therefore, scientists at the Center for Medical, Agricultural and Veterinary Entomology have purified, characterized and cloned the first glutathione transferase in the order Hymenoptera. Understanding insecticide disposition in fire ants is crucial to the development of insect-specific insecticides.
Technical 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 mol/min/mg, 0.13 mM, 0.84 mM, 74.5 sec-1, 573.1 mM-1 sec-1, and 88.7 mM-1 sec-1, respectively. An internal fragment of the enzyme, released by endoproteinase Lys-C digestion, was sequenced and used to design a degenerate primer. Purified cDNA from a -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 comprised of 202 amino acids with a calculated molecular weight of 23433 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 to 258, and 457 to 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. Keywords: Solenopsis invicta; Glutathione S-transferase; RT-PCR; RACE; cDNA cloning