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Purification and Characterization of Naturally Soluble Neuropathy Target Esterase from Chicken Sciatic Nerve by HPLC and Western Blot
Author(s) -
Escudero M. A.,
Vilanova E.
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.69051975.x
Subject(s) - biotinylation , chemistry , western blot , blot , esterase , paraoxon , alkaline phosphatase , biochemistry , chromatography , size exclusion chromatography , phosphatase , molecular mass , microbiology and biotechnology , enzyme , acetylcholinesterase , biology , gene
Neuropathy target esterase (NTE) activity is defined operatively as the paraoxon‐resistant mipafox‐sensitive phenyl valerate esterase activity. A preparation containing a soluble isoform (S‐NTE2) has been obtained from sciatic nerve. It was inhibited by the biotinylated organophosphorous ester S9B [1‐(saligenin cyclic phospho)‐9‐biotinyldiaminononane] in a progressive manner showing a second‐order rate constant of (3.50 ± 0.26) × 10 6 M −1 · min −1 with an I 50 for 30 min of 6.6 ± 0.4 n M . S‐NTE2 was enriched 218‐fold by gel filtration followed by strong and weak anion‐exchange chromatographies in HPLC. In western blots, this enriched sample showed two bands of endogenous biotinylated polypeptides after treating the blots with streptavidin‐alkaline phosphatase complex. When the sample was treated with S9B, another biotinylated band was observed with a molecular mass of ∼56 kDa, which was not seen when the sample had been pretreated with mipafox before the S9B labeling. It was deduced that this band represents a polypeptide (identified as the S‐NTE2 protein) that is bound by both mipafox and S9B and that should be responsible for the progressive S9B inhibition. It is possible that S‐NTE2 is the target for attack by compounds that promote delayed neuropathy.