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

Abstract: 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 (∼ 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. The titration curve corresponding to inhibition by mipafox was studied over the 0.1–200 μM range, in the presence of 40 μM paraoxon, and data obtained were fitted to models of one or two exponential mipafox‐sensitive components plus a resistant component. Mipafox‐resistant activity was 38 and 52% of total paraoxon‐resistant activity for the particulate and soluble fractions, respectively. Particle data suggest that P‐NTE contains mainly one component with I50 of ∼5.4–7.3 μM, this representing >85% of total mipafox‐sensitive activity. However, the soluble fraction data fit better to two sensitive components: high‐ and low‐mipafox‐sensitive components with I50 of 4.9 and 43 μM, representing 35 and 65% of total paraoxon‐resistant activity, respectively.

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