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United States Department of Agriculture

Agricultural Research Service

Title: Purification and Characterization of Trans-Permethrin Metabolizing Microsomal Esterases from Workers of the Eastern Subterranean Termites, Reticulitermes Flavipes (Kollar)

Authors
item Valles, Steven
item Oi, Faith
item Strong, Charles

Submitted to: Journal of Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 9, 2000
Publication Date: N/A

Interpretive Summary: Subterranean termite damage and control efforts cost Americans an estimated 2 billion dollars annually. Although insecticides are the primary method of controlling subterranean termites, very little research has been conducted on their detoxification systems. Previous studies have shown that permethrin (a common termiticide currently in use in the United States) was metabolized by the eastern subterranean termite hydrolytically In an effort to more fully understand the detoxification processes in termites, scientists at the Center for Medical, Agricultural and Veterinary Entomology have purified and characterized three of the most active permethrin metabolizing esterases from the eastern subterranean termite. This research provides crucial basic knowledge of xenobiotic detoxification in termites and serves as a starting point for future research with termite detoxification systems.

Technical Abstract: Three naphthyl acetate hydrolyzing esterase isozymes were purified from microsomes prepared from Reticulitermes flavipes workers. The two step process involved sequential preparative IEF followed by continuous elution preparative electrophoresis on a 5% non-denaturing polyacrylamide gel. The first IEF run resulted in 5.4-fold purification with a yield of 46.1%. Subsequent IEF further purified the esterases 14.3-fold and 12% yield. Preparative electrophoresis of the pooled IEF fractions produced 3 major peaks of naphthyl acetate hydrolyzing activity. The esterases were correspondingly designated microsomal esterase (ME) 1, ME 2, and ME 3 based on increasing molecular retention on a native PAGE gel. ME 1, ME 2, and ME 3 were acidic proteins with pI values of 4.61, 4.70, and 4.77, respectively. Molecular mass as determined by gel filtration chromatography of ME 1, ME 2, and ME 3 was 69, 64, and 62 kDa, respectively. SDS-PAGE gels produced a single band for each of the isozymes with a molecular mass of 63 kDa indicating that the esterases were monomers. Specific activities of ME 1, ME 2, and ME 3 increased with increasing pH and the enzymes were active over a broad temperature range (25 to 55oC). The three purified isozymes were inhibited at low concentration by paraoxon, chlorpyrifos, DEF, and PMSF indicating that they were "B" type serine esterases. None of the isozymes was inhibited by 10-4 M imidacloprid, fipronil, or PBO . Quantitatively, ME 1, ME 2 and ME 3 metabolized trans-permethrin at 21.8, 21.0, and 38.8 nmol/hour/mg protein, representing a purification factor of 333-, 318-, and 591-fold over microsomes, respectively. The 3 isozymes produced the same type and number of trans-permethrin metabolites.

Last Modified: 9/10/2014
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