| Organophosphate Induced Delayed Neuropathy is characterized by degeneration of the distal ends of the long, large diameter nerves of the peripheral and central nervous system. This degeneration is induced by exposure to certain organophosphates, such as diisopropylfluorophosphate (DFP) and Mipafox, but not by others, such as Paraoxon. Two potential target sites for the induction of OPIDN have been proposed. The first is a protein with an apparent molecular weight on SDS-PAGE of 160,000 which can be radiolabeled with ({dollar}sp3{dollar}H) DFP in a Mipafox sensitive but Paraoxon insensitive manner. The second is a membrane associated phenyl valerate hydrolase activity known as Neuropathy Target Esterase (NTE) which is inhibited by Mipafox but not by Paraoxon. My initial experiments showed that NTE, isolated from chick-embryo brain membranes, migrates as a single peak on sucrose gradients and by gel filtration. Analysis by SDS-PAGE determined that the M{dollar}sb{lcub}rm r{rcub}{dollar} 160,000 ({dollar}sp3{dollar}H) DFP labeled protein co-migrated on sucrose gradients with active NTE, thereby indicating that they have similar physical characteristics. Optimal stabilization, storage and solubilization conditions were then developed for NTE. Triton X-100, 0.5 M NaCl solubilized NTE was purified by gel filtration, ion exchange chromatography and sucrose density gradient centrifugation, but only gel filtration yielded a sample with a higher specific activity than the starting sample.; The topography of the catalytic site of NTE was then examined by investigating the inhibition of NTE by a series of 3-alkylthio- and 3-arylthio-1,1,1-propan-2-ones, which were found to be rapidly reversible, competitive inhibitors of NTE with I{dollar}sb{lcub}50{rcub}{dollar} values between 1.3 {dollar}times{dollar} 10{dollar}sp{lcub}-4{rcub}{dollar} M to 4.9 {dollar}times{dollar} 10{dollar}sp{lcub}-8{rcub}{dollar} M. Correlation of I{dollar}sb{lcub}50{rcub}{dollar} values with octanol/water partition coefficients (P) found that the optimal lipophilicity for NTE substrates and inhibitors was in the range of logP = 3.0 to 3.4. These results indicated that a large hydrophobic pocket is closely associated with the catalytic residue of NTE. An affinity resin was prepared by attachment of 3-(9{dollar}spprime{dollar}-mercaptononylthio)-1,1,1-trifluoropropan-2-one to epoxy-activated Sepharose CL4B. Typically greater than 80% of the NTE activity in samples was bound to this affinity resin while {dollar}<{dollar}1% of the total protein in samples was bound. Binding was blocked by Mipafox but not Paraoxon. This method in combination with preparative SDS-PAGE therefore provides a means to rapidly purify microgram quantities of NTE for sequencing and further biochemical characterization. |
