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 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 three major peaks of alpha-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 (25-55 degrees C). The three purified isozymes were inhibited at low concentration by paraoxon (10(-10) M), chlorpyrifos (10(-6) M), DEF (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, PHMB, or CaCl(2), further supporting the conclusion that the microsomal esterases were of the "B" type. None of the isozymes was inhibited by 10(-4) M imidacloprid, fipronil, or PBO. Quantitatively, ME 1, ME 2 and ME 3 metabolized t-permethrin at 21.8, 21.0, and 38.8 nmol/h/mg protein, representing a purification factor of 333-, 318-, and 591-fold over microsomes, respectively. The three isozymes produced the same type and number of t-permethrin metabolites.

Alpha-naphthyl acetate esterase-1 (ANAE-1) secreted by epithelioid cells from induced rabbit lung granuloma showed MIF activity.

The function of alpha-naphthyl acetate esterase-1, whose isoelectric point values range from 5.15 to 5.45, was examined. A higher value of alpha-naphthyl acetate esterase-1 was detected in the extracts of epithelioid cells isolated from rabbit lung granuloma at 4 weeks after injection of Freund's complete adjuvant, compared to those values of alveolar macrophages isolated from the same lungs described above and of the normal lungs. Additionally, this enzyme activity was observed to be prominent in the culture supernatants of epithelioid cells. alpha-Naphthyl acetate esterase-1 was purified from lung granuloma as a single 62-kDa band by SDS-PAGE analysis. The purified enzyme showed a macrophage migration inhibition activity at concentrations over 20 nM, and its activity was dose-dependent. Moreover, when various amounts of the purified enzyme were added to lymphocyte-derived macrophage migration inhibitory factor, macrophage migration inhibition was significantly enhanced with a dose-dependent manner. The results suggest that alpha-naphthyl acetate esterase-1 secreted by granuloma macrophages, particularly by epithelioid cells, contributes to granuloma formation.

Two-dimensional separation of alpha-naphthyl acetate esterases in human leucocytes and platelets.

Normal human leucocytes and platelets contain esterases which hydrolyse alpha-naphthyl acetate (alpha NA). Purified preparations from these cells were investigated by isoelectric focusing and subsequent polyacrylamide gradient gel electrophoresis at pH 9.0. Extractable alpha NA esterases were separated according to isoelectric point (pI) and molecular weight (MW). Monocytes, lymphocytes, granulocytes and platelets contain a unique pattern of alpha NA esterases, most of which can be inhibited by diisopropyl fluorophosphate (DFP; 0.1 mM). Their activity, however, is not affected by eserine (0.1 mM) or p-hydroxymercuribenzoate (1 mM). No protease activity of these enzymes was detected; it is likely that the majority constitute carboxylesterases (EC 3.1.1.1). Monocytes contain five alpha NA esterases which are additionally inhibited by bis(4-nitrophenyl)-phosphate (0.1 mM) and sodium fluoride (40 mM). PIs are in the range 5.7-6.2 and MWs are 145 000, 155 000, 250 000, 290 000 and 340 000. These enzymes are specific for monocytes. Platelets are characterized by a group of alpha NA esterases having pIs between 6.5 and 8.0, these corresponding to MWs ranging from 15 000 to 400 000.

Fractionation and further characterization of granulocytic and monocytic alpha-naphthyl acetate (ANAE) esterases.

Following characterization of myeloid nonspecific esterases by isoelectric focusing (IEF), two main groups of alpha-naphthyl acetate (ANAE) esterase isoenzymes were defined and fractionated from cytoplasmic extracts by chromato focusing techniques according to differences in their isoelectric points (pI). The first of these ANAE enzyme groups was common to leukocytes of both granulocytic and monocytic lineage, while the other, which characteristically comprised a group of isoenzymes within the pI range 5.5-6.1, was specifically associated with monocytic differentiation. The properties of the two purified ANAE enzyme fractions were compared by inhibition (heat and sodium fluoride) and further electrophoretic studies, and the results discussed in relation to the cytochemical characterization of these enzymes as markers of specific myeloid cell differentiation.

[Characterization of Escherichia coli esterase B after separation by chromatography]. / Caractérisation de l'estérase B d'Escherichia coli après séparation par chromatographie.

Esterase B of Escherichia coli has been purified 56 fold with recovery of 39%. The apparent molecular weight as determined by gel filtration was approximately 57000. The pI as determined by isoelectric focusing was 4.6. This enzyme exhibited Michaelis-Menton kinetics with apparent Km of 0.25 mM for l-naphtyl acetate. It remained stable at 60 degrees C but was sensitive to pH values below 6. The esterase activity was completely inhibited by Di-isopropyl-fluorophosphate (DFP) but was resistant to iodoacetamide and to EDTA.

[Detection of alpha-naphthyl acetate esterase in Francisella tularensis]. / Obnaruzhenie al'fa-naftilatsetatésterazy u Francisella tularensis.

The activity of alpha-naphthyl-acetate esterase (alpha-NAE) has been studied in Francisella tularensis strains belonging to 3 subspecies. Alpha-NAE has been detected spectrophotometrically in native cells and cell extracts of the virulent nonarctic strains Schu and Cole, holarctic virulent strain No. 503/841 and holarctic vaccine strain No. 15/10, Central Asian virulent strain No. 543, as well as their attenuated variants. In all the strains studied the presence of 7 alpha-NAE isoenzymes has been established by means of electrophoresis in acrylamide gel, and the relative electrophoretic activity and the enzymatic activity of these isoenzymes have been determined. The nonarctic and holarctic strains have been found to differ from the Central Asian strain in the activity of 2 alpha-NAE isoenzymes, named characteristic isoenzymes. After the attenuation of the strains belonging to all subspecies the enzymatic composition of these strains remained unchanged, but at the same time the total activity of the holarctic and Central Asian strains increased, while that of the nonarctic strains decreased. These differences in the activity of alpha-NAE isoenzymes, if confirmed in further studies on a greater number of strains belonging to 3 Francisella tularensis subspecies, can be used as an additional sign for differentiation.