Purification and characterization of trans-permethrin metabolizing microsomal esterases from workers of the eastern subterranean termite, Reticulitermes flavipes (Kollar)
Sm. Valles et al., Purification and characterization of trans-permethrin metabolizing microsomal esterases from workers of the eastern subterranean termite, Reticulitermes flavipes (Kollar), INSEC BIO M, 31(6-7), 2001, pp. 715-725
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.