NTE soluble isoforms: new perspectives for targets of neuropathy inducers and promoters

Neural carboxylesterases can be discriminated by differential inhibition as says with organophosphorus compounds (OPs), paraoxon (O,O'-diethyl p-nitrop henyl phosphate) and mipafox (N,N'-diisopropyl phosphorodiamidofluoridate) being the ones used to discriminate esterases that should be either irrelev ant or candidates as targets of the mechanism of induction of the organopho sphorus-induced delayed polyneuropathy (OPIDP). The brain membrane-bound ph enyl valerate esterase (PVase) defined by Dr Johnson in 1969 as neuropathy target esterase (NTE) and recently cloned by Dr Glynn and coworkers is term ed here as particulate NTE due to its association to the membrane particula te fraction. It is considered as the target of OPIDP and is the activity me asured in standard NTE assays and toxicity tests. Following the same operat ional criteria in the soluble fraction of sciatic nerve a paraoxon-resistan t but mipafox-sensitive PVase activity was described and termed as S-NTE, w ith an apparent lower sensitivity to some inhibitors than particulate NTE. Two isoforms (S-NTE1 and S-NTE2) were subsequently separated by gel filtrat ion chromatography. In a partly purified S-NTE2 preparation polypeptides we re identified in western blots by labelling with S9B [1-(saligenin cyclic p hospho)-9-biotinyldiaminononane], the same biotinylated OP used to label an d isolate particulate NTE, but not with anti-particulate NTE antibodies. Fr om sequential inhibition protocols, inhibitor washing-out and time course i nhibition Studies it is deduced that reversibility of inhibition is a new f actor introducing a higher complexity in the identification of the esterase s that could be candidates as targets of the mechanisms of induction and/or promotion of neuropathy. We have evidences that in sciatic nerve soluble f raction a high proportion (about 70%) of the activity that is inhibited by paraoxon in the usual concurrent assay is quickly reactivated after removin g paraoxon and it is permanently inhibited by mipafox. Under this improved sequential paraoxon/mipafox inhibition procedure S-NTE represents about 50% of total PVases while in the usual concurrent assay it was only apparently about 1-2%. Moreover with such criteria, S-NTE2 isoform(s) represents abou t 97-99% of total S-NTE, and S-NTE1 is only a marginal amount probably resu lting of a partial solubilization from particulate NTE. Fixed time inhibito n curves with variable mipafox concentration failed to discriminate more th an one component. However kinetic behaviour of the time progressive inhibit ion cannot be explained by a simple model with a single exponential mathema tical component, indicating that either the possibility of more than one co mponent or a more complex mechanistic model should be considered. Consequen tly both particulate NTE and S-NTE assay protocols and their role in induct ion and promotion of neuropathies will need to be reviewed. Data published by Drs Lotti, Moretto and coworkers suggest that particulate NTE cannot be the target of promotion of axonopathies. The proposal that S-NTE2 could be such a target is suggestive and under collaborative biochemical and toxicol ogical studies. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.