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 µ M paraoxon but sensitive to 250 µ M mipafox. 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 corresponding to inhibition by mipafox was studied over the 1–250 µ M range, in the presence of 40 µ M paraoxon. The data revealed that S-NTE1 and S-NTE2 have different sensitivities to mipafox with I₅₀ (30 min) values of 1.7 and 19 µ M , respectively. This was similar to the pattern observed in the soluble fraction from sciatic nerve with two components ( V _o 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-molecular-mass (>700-kDa) peak or S-NTE1 was obtained. It showed an I₅₀ of 5.2 µ M 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 V _o peak activity decreased in the soluble fraction from these nerve tissues. This suggests that the V _o peak could be related to materials partly solubilized from membranes at higher pH. The chromatographic pattern 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.     

Soluble and Particulate Organophosphorus Neuropathy Target Esterase in Brain and Sciatic Nerve of the Hen, Cat, Rat, and Chick

Considerable evidence exists suggesting that the so-called neuropathy target esterase (NTE) is involved in the mechanisms responsible for organophosphorus-induced delayed polyneuropathy (OPIDP). Earlier studies in the adult hen, the habitually employed experimental model in OPIDP, have shown that most NTE activity in the brain is centered in paniculate fractions, whereas approximately 50% of this activity in the sciatic nerve is encountered in soluble form, with the rest being paniculate NTE. In the present work, we have studied the paniculate and soluble fractional distribution of paraoxon-resistant phenylvalerate esterase activity (B activity), parabxon- and mipafox-resistant phenylvalerate esterase activity (C activity), and NTE activity (B - C) according to ultracentrifugation criteria (100,000 g for 1 h). To this effect, two sensitive (adult hen and cat) and two scarcely sensitive (rat and chick) models were used. In all four experimental models, the distribution pattern was qualitatively similar: B activity and total NTE were much greater in brain (900–2, 300 nmol/min/g of tissue) than in sciatic nerve (50–100 nmol/min/g of tissue). The proportion of soluble NTE in brain was very low (<2%), whereas its presence in sciatic nerve was substantial (30–50%). The NTE/B ratio in brain was high for the particulate fraction (>60%) and low in the soluble fraction (7–30%); in sciatic nerve the ratio was about 50% in both fractions. Slight quantitative differences were observed in terms of OPIDP sensitivity: the proportion of soluble NTE in sciatic nerve was slightly higher in the sensitive animals (hen and cat: 49 and 44%, respectively) than in the rat and chick (41 and 37%, respectively), although no differences were noted in terms of concentration (in nanomoles per minute per gram of tissue). It is concluded that the distribution pattern of the activities studied is similar in all four experimental models, with no important quantitative differences directly related to species sensitivity or age.     

Soluble and Participate Forms of the Organophosphorus Neuropathy Target Esterase in Hen Sciatic Nerve

Neuropathy target esterase (NTE) is the suggested "target" molecule involved in the initiation of organophosphorus-induced delayed polyneuropathy. Sciatic nerve NTE was separated into particulate (P-NTE) and soluble (S-NTE) fractions by ultracentrifugation at 100,000 g for 1 h in 0.32 M sucrose and compared with the corresponding brain extract. Total sciatic NTE activity was 80-100 nmol/min/g tissue from which 50-60% was recovered in the soluble supernatant fraction and the remaining 40-50% in the pellet fraction. About 90% of brain tissue activity (approximately 1,800 nmol/min/g tissue) was recovered as P-NTE. A similar distribution was obtained when more drastic centrifugation without sucrose was performed. P-NTE and S-NTE were distributed with the membrane and cytosolic markers assayed, respectively, glucose-6-phosphatase, Na+,K(+)-ATPase, 5'-nucleotidase, phospholipids, and lactate dehydrogenase. When the pH during the centrifugation was increased from 6.4 to 11, recovered P-NTE activity decreased from 1,750 to 118 nmol/min/g tissue for brain and from 31 to 12 nmol/min/g for sciatic nerve. However, S-NTE activity and total nonfractionated control activity were only slightly affected by the same pH treatment. The distribution pattern encountered may be better understood as representing two different proteins than an equilibrium between soluble and membrane-bound portions of a single protein, with P-NTE activity depending on a membrane factor from which it is separated through fractionation at high pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

Abstract: 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) × 10⁶ M ⁻¹· min⁻¹ with an I₅₀ 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.     

Neuropathy Target Esterase of Hen Brain: Active Site Reactions with 2-[octyl-3H]Octyl-4H-1,3,2-Benzodioxaphosphorin 2-Oxide and 2-Octyl-4H-1,3,2-[aryl-3H] Benzodioxaphosphorin 2-Oxide

Abstract: 2-Octyl-4H-1,3,2-benzodioxaphosphorin 2-oxide (octyl-BDPO) is one of the most potent inhibitors known for neuropathy target esterase (NTE) of hen brain with 50% inhibition at 0.2 nM. Two NTE-like proteins, i.e., resistant to paraoxon and sensitive to mipafox, of ～155 and ～119 kDa (designated NTE-155 and NTE-119, respectively) are labeled by [octyl-³H]octyl-BDPO and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling with [aryl-³H]octyl-BDPO is only ～15% of that with [octyl-³H]octyl-BDPO, indicating that the majority of the phosphorylated NTE undergoes aging with only a small proportion of nonaged target or intramolecular group transfer (“alkylation”). NTE-155 and NTE-119 have the same kinetic constants and maximal number of phosphorylation sites, equivalent for each of them to 26 fmol/mg of protein and totalling at least 0.44–1.2 µg of NTE protein/g of brain. Structure-activity investigations involving 17 combinations of organophosphorus (OP) compounds of varied chemical type, stereo-chemistry, and concentration establish an excellent correlation (r = 0.95) between inhibition of NTE activity and protein labeling and thereby the toxicological relevance of these assays, which equally implicate NTE-155 and NTE-119 (probably an autolysis product of NTE-155) as targets in OP-induced delayed neuropathy. [octyl-³H]-Octyl-BDPO is an improved probe for NTE in terms of its potency, reactivity, selectivity, and the formation of ³H-labeled NTE with a stable phosphorus-carbon bond.     

Modeling the Tertiary Structure of the Patatin Domain of Neuropathy Target Esterase

Neuropathy target esterase (NTE) is a transmembrane protein of unknown function whose specific chemical modification by certain organophosphorus (OP) compounds leads to distal axonopathy. Therefore, solving the 3D structure of NTE would advance the understanding of its pathogenic and physiologic roles. In this study, the tertiary structures of the patatin (catalytic) domain and the N-terminal transmembrane domain of NTE were modeled using the crystal structures of patatin (PDB ID 1oxw) and moricin (PDB ID 1kv4) as templates. Sequence alignments and secondary structure predictions were obtained from the INUB server (Buffalo, NY). O and PyMol were used to build the PNTE and NTE TMD chains from these sequence alignments. The PNTE model was refined in the presence of water using the crystallography and NMR system, while the NTE TMD model was refined in vacuo using the GROMOS implementation in the Swiss PDB viewer. The modeled active site of NTE was found to consist of a Ser966-Asp1086 catalytic dyad, which is characteristic of phospholipase A2 enzymes. The Ser966 Ogamma was located 2.93 A from the Odelta2 of Asp1086. In addition, our NTE model was found to contain a single N-terminal transmembrane domain. This modeling effort provided structural and mechanistic predictions about the catalytic domain of NTE that are being verified via experimental techniques.     

Purification of Neuropathy Target Esterase from Avian Brain After Prelabelling with [3H]Diisopropyl Phosphorofluoridate

Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The volatilizable counts assay and analytical SDS-PAGE were used to monitor the protein. The 150-kDa subunit polypeptide appears as a single band on analytical SDS-PAGE.     

Species Distribution of Paraoxon-Resistant Brain Polypeptides Radiolabeled with Diisopropyl Phosphorofluoridate ([3H]DiPF): Electrophoretic Assay for the Aged Polypeptide of [3H]DiPF-Labelled Neuropathy Target Esterase

Brain neuropathy target esterase is identified as a paraoxon-resistant, mipafox-sensitive esterase that can be labelled with [3H]diisopropyl phosphorofluoridate. During "aging" of the labelled (inhibited) esterase, half the label (one isopropyl group) is transferred to a site (of the same molecular weight in sodium dodecyl sulphate) whence it may be released in volatile form by treatment with alkali. Our previously published procedure for complete extraction in a form suitable for scintillation counting of tritium-labelled proteins from polyacrylamide gels includes treatment of part-solubilised gels with alkali. Particles from brain of the hen, pig, sheep, guinea-pig, and rat were preincubated with paraoxon with or without mipafox, treated with [3H]diisopropyl phosphorofluoridate, and solubilised in sodium dodecyl sulphate. Labelled polypeptides (except from the rat) were separated by electrophoresis. Both mipafox-sensitive labelling and "volatilisable counts" were located principally in the 155-kilodalton region, with the residues dispersed throughout the gels. The quantities of paraoxon-resistant, mipafox-sensitive labelling sites and of "volatilisable counts" (in pmol/particles from 1 g) were, respectively, 12.2 and 8.65 in hen brain, 9.80 and 6.82 in pig, 8.48 and 5.46 in sheep, 4.46 and 4.01 in guinea-pig, and 4.91 and 2.08 in rat. The "volatilisable count" assay seems more specific for neuropathy target esterase and is easier and more precise than assays based on differences in labelling of two samples, each subjected to much processing. Hydrolytic activity of particles taken before labelling was measured against phenyl valerate.(ABSTRACT TRUNCATED AT 250 WORDS)