Separation of two forms of neuropathy target esterase in the soluble fraction of the hen sciatic nerve

Escudero, M.A.; Barril, J.; Tormo, N.; Vilanova, Eugenio

doi:10.1016/0009-2797(95)03620-2

Cited by 9 publications

(9 citation statements)

References 13 publications

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“…Although two apparent soluble NTE froms, S-NTE1 and S-NTE2, were observed when tissue was prepared at pH 8 (see introductory section), only S-NTE2 was obtained in the preparations at pH 6.8, used in the present study, representing the actually soluble NTE form (Escudero et al, 1995(Escudero et al, , 1996. This study shows that S-NTE2 is inhibited by biotinylated OP S9B in a time-progressive manner, which makes it a good S-NTE2 labeling ligand.…”

Section: Discussionmentioning

confidence: 79%

“…Tissue preparation and subcellular fractionation for preparing the soluble fraction (100,000 g supernatant) from sciatic nerve were performed as previously described (Escudero et al, 1995), except that the buffer for homogenization and centrifugation in separating the soluble fraction was 10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 1 mM EDTA, and 0.32 M sucrose with pH adjusted to 6.8.…”

Section: Tissue Preparationmentioning

confidence: 99%

“…Twelve samples of 5 ml (total volume, 60 ml) containing the soluble fraction (from 200mg of original tissue/ml) from 12 g of fresh sciatic nerve tissue were eluted using Tris buffer, pH 8.0, as previously described (Escudero et al, 1995). After measuring the activities and protein, the subfractions containing the highest specific activity (96-110 ml elution volume) from the 12 chromatograms (150 ml) were pooled and concentrated to 3.6 ml by ultrafiltration.…”

Section: Preparation Of Partly Purified Enriched S-nte2 Samplementioning

confidence: 99%

“…However, from the mipafox titration curve in the presence of 40 ,uM paraoxon, it was predicted that S-NTE contains two components of different sensitivity (I5~= 4.9 and 43 tM) (Vilanova et al, 1990). This was later confirmed when both components were separated into two peaks by molecular exclusion chromatography in Sephacryl S-300 (Escudero et al, 1995). These were designated as V 0 and lOOK peaks, and we are presently calling the NTE activity contained in these peaks S-NTE1 and S-NTE2, respectively.…”

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confidence: 99%

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

Escudero

Vilanova

1997

Journal of Neurochemistry

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Neuropathy target esterase (NTE) activity is defined operatively as the paraoxon-resistant mipafoxsensitive 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-(saligenincyclic phospho)-9-biotinyldiaminononane] in a progressive manner showing a second-order rate constant of (3.50 ±0.26) x 106 M 1 min~1with an l5for 30 mm 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 -~56kDa, 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. Key Words: Neuropathy target esteraseOrganophosphorus-Soluble esterase-Sciatic nerve.

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Section: Discussionmentioning

confidence: 79%

Section: Tissue Preparationmentioning

confidence: 99%

Section: Preparation Of Partly Purified Enriched S-nte2 Samplementioning

confidence: 99%

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confidence: 99%

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

Escudero

Vilanova

1997

Journal of Neurochemistry

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“…12,22,18,23,24 It has been suggested that the target of promotion is embraced in a fraction which has been separated by molecular exclusion chromatography of the soluble fraction from the chick sciatic nerve called peak V 0 . 25,26 This fraction was first described by Escudero and co-workers in soluble esterases of the brain, spinal cord, and sciatic nerve. 26,27 The chicken is the animal model of current use specified by regulatory agencies for the evaluation of the delayed neuropathic potential of organophosphorus esters in humans (OECD 418 and 419 testing guide).…”

Section: Introductionmentioning

confidence: 96%

Phenylmethylsulfonyl Fluoride, a Potentiator of Neuropathy, Alters the Interaction of Organophosphorus Compounds with Soluble Brain Esterases

Mangas

Vilanova

Estévez

2012

Chem. Res. Toxicol.

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Phenylmethylsulfonyl fluoride (PMSF) is a protease and esterase inhibitor that causes protection or potentiation/promotion of organophosphorus delayed neuropathy (OPIDN) depending on whether it is dosed before or after an inducer of delayed neuropathy. The molecular target of promotion has not yet been identified. Kinetic data of esterase inhibition were first obtained for PMSF with a soluble chicken brain fraction and then analyzed using a kinetic model with a multienzymatic system in which inhibition occurred with the simultaneous chemical hydrolysis of the inhibitor and ongoing inhibition (inhibition during the substrate reaction). The best fitting model was a model with resistant fraction, Eα (28%), and two sensitive enzymatic entities, Eβ (61%) and Eγ (11%), with I(50) at 20 min of 70 and 447 μM, respectively. The estimated constant of the chemical hydrolysis of PMSF was kh = 0.23 min(-1). Eα, which is sensitive to mipafox and resistant to PMSF, became less sensitive to mipafox when the preparation was preincubated with PMSF. Its Eα I(50) (30 min) of mipafox increased with the PMSF concentration used to preincubate it. Eγ is sensitive to both PMSF and mipafox, and after preincubation with PMSF, Eγ became less sensitive to mipafox and was totally resistant after preincubation with 10 μM PMSF or more. The sensitivity of Eα to paraoxon (I(50) 30 min from 9 to 11 nM) diminished after PMSF preincubation (I(50) 30 min 185 nM) and showed no spontaneous reactivation capacity. The nature of these interactions is unknown but might be due to covalent binding at sites other than the substrate catalytic center. Such interactions should be considered to interpret the potentiation/promotion phenomenon of PMSF and to understand the effects of multiple exposures to chemicals.

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Soluble phenyl valerate esterases of hen sciatic nerve and the potentiation of organophosphate induced delayed polyneuropathy

Gambalunga

Pasqualato

Lotti

2010

Chemico-Biological Interactions

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Separation of two forms of neuropathy target esterase in the soluble fraction of the hen sciatic nerve

Cited by 9 publications

References 13 publications

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

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

Phenylmethylsulfonyl Fluoride, a Potentiator of Neuropathy, Alters the Interaction of Organophosphorus Compounds with Soluble Brain Esterases

Soluble phenyl valerate esterases of hen sciatic nerve and the potentiation of organophosphate induced delayed polyneuropathy

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