Purification and characterization of naturally soluble neuropathy target esterase from chicken sciatic nerve by HPLC and western blot.
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) x 10(6) M(-1) x min(-1) with an I50 for 30 min of 6.6 +/- 0.4 nM. 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 approximately 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.
Escudero
M A
MA
Department of Neurochemistry, Institute of Neurosciences, University of Alicante, Campus of San Juan, Spain.
Vilanova
E
E
D000818:Animals; D015415:Biomarkers; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D012584:Sciatic Nerve / Q000201:enzymology*
D015415:Biomarkers; D002265:Carboxylic Ester Hydrolases; C021859:neurotoxic esterase