Purification and characterization of trans-permethrin metabolizing microsomal esterases from workers of the eastern subterranean termite, Reticulitermes flavipes (Kollar)

Three alpha -naphthyl acetate hydrolyzing esterase isozymes were purified f rom 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%. Subs equent IEF further purified the esterases 14.3-fold and 12% yield. Preparat ive electrophoresis of the pooled IEF fractions produced three major peaks of alpha -naphthyl acetate hydrolyzing activity. The esterases were corresp ondingly designated microsomal esterase (ME) 1, ME 2, and ME 3 based on inc reasing 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. Mole cular mass as determined by gel filtration chromatography of ME 1, ME 2, an d ME 3 was 69, 64, and 62 kDa, respectively. SDS-PAGE gels produced a singl e 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 te mperature range (25-55 degreesC). The three purified isozymes were inhibite d at low concentration by paraoxon (10(-10) M), chlorpyrifos (10(-6) M), DE F (10(-6) M), and PMSF (10(-6) M) indicating that they were "B" type serine esterases. Conversely, inhibition was not observed at 10(-4) M eserine, PH MB, or CaCl2, further supporting the conclusion that the microsomal esteras es were of the "B" type. None of the isozymes was inhibited by 10(-4) M imi dacloprid, fipronil, or PBO. Quantitatively, ME I,ME 2 and ME 3 metabolized t-permethrin at 21.8, 21.0, and 38.8 nmol/h/mg protein, representing a pur ification factor of 333-, 318-, and 591-fold over microsomes, respectively. The three isozymes produced the same type and number of t-permethrin metab olites. Published by Elsevier Science Ltd.