Chromatographic Discrimination of Soluble Neuropathy Target Esterase Isoenzymes and Related Phenyl Valerate Esterases from Chicken Brain, Spinal Cord, and Sciatic Nerve

Abstract

Neuropathy target esterase (NTE) activity is operatively defined in this work as the phenyl valerate esterase (PVase) activity resistant to 40 ,aM paraoxon but sensitive to 250~eMmipafox. Gel filtration chromatography with Sephacryl S-300 of the soluble fraction from spinal cord showed two PVase peaks containing NTE activity (S-NTE1 and S-NTE2). The titration curve corre-.sponding to inhibition by mipafox was studied over the 1 -250 ,uM range, in the presence of 40 ,..tM paraoxon. The data revealed that S-NTE1 and S-NTE2 have different sensitivities to mipafox with l5~(30 mm) values of 1.7 and 19~.eM,respectively. This was similar to the pattern observed in the soluble fraction from sciatic nerve with two components (V 0 peak, or S-NTE1; and 100-K peak, or S-NTE2) with different sensitivity to mipafox. However, in the brain soluble fraction, only the high-molecularmass (>700-kDa) peak or S-NTE1 was obtained. It showed an 150 of 5.2~eM in the mipafox inhibition curve. The chromatographic profile was different on changing the pH in the subcellular fractionation. When the homogenized tissue was centrifuged at pH 6.8, the V0 peak activity decreased in the soluble fraction from these nerve tissues. This suggests that the V0 peak could be related to materials partly solubilized from membranes at higher pH. The chrom~atographicpattern and mipafox sensitivity suggest that the different tissues have a different NTE isoform composition. S-NTE2 should be a different entity than S-NTE1 and particulate NTE. The potential role of soluble forms in the mechanism of initiation or promotion of neuropathy due to organophosphorus remain unknown. Key Words: Organophosphorus-Phenyl valerate esterase-Neuropathy target esterase. compound-induced delayed polyneuropathy; P-NTE and S-NTE, particulate and soluble neuropathy target esterase, respectively; PV, phenyl valerate; PVase, phenyl valerate esterase.

Kinetics of inhibition of soluble peripheral nerve esterases by PMSF: a non-stable compound that potentiates the organophosphorus-induced delayed neurotoxicity

“…Both sensitive components (EI and EII) showed spontaneous reactivation after the inhibition with paraoxon (Estévez et al 2011), EI is permanently inhibited with S9B, and EII is spontaneously reactivated after the inhibition with S9B (Estévez et al 2010). The component EI might be related to the so-called soluble NTE (Escudero et al 1997) because it is permanently inhibited with mipafox, S9B and PMSF but it is spontaneously reactivated after the inhibition by paraoxon.…”

Loss of neuropathy target esterase in mice links organophosphate exposure to hyperactivity

“…The function of NTE-R1 is unknown, although it may be related to esterase activities in different fractions of hen brain and sciatic nerves [30][31][32] . Our results clearly show that Nte-R1 cannot substitute for Nte during embryonic development and that it is not responsible for the effects of EOPF.…”

Purification and Characterization of Naturally Soluble Neuropathy Target Esterase from Chicken Sciatic Nerve by HPLC and Western Blot

“…Nevertheless, the possibility that S-NTE2 could be a proteolytic product of P-NTE is unlikely; the incubation of brain membranes (containing large amounts of P-NTE) with sciatic nerve homogenates did not increase the NTE recovered in soluble fractions (data not shown). However, from previously published observations on S-NTE separation of S-NTEI and S-NTE2 (Escudero et al, 1997), it was deduced that S-NTE1 could constitute membrane material that can be solubilized at pH 8 but not at lower pH values. As the enriched preparation of S-NTE2 comes from a peak in Sephacryl S-300 of~100 kDa molecular mass, the native protein is likely to be a dimer.…”

“…Thus, S-NTE1 and S-NTE2 can be discriminated by both physical chromatographic separation and by sensitivity to mipafox. It was subsequently observed in several tissues that S-NTE1 is a form detected in soluble fraction prepared at pH 8.0 (Escudero et al, 1997). That S-NTE1 is not obtained at pH 6.8 and that its sensitivity to mipafox is similar to that of P-NTE suggest that it is a membrane component partly solubilized at higher pH values and is derived either from P-NTE or from some other, similar membrane protein.…”