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Identification and isolation of a 155-kDa protein with neuropathy target esterase activity
C E Mackay1, B D Hammock, B W Wilson
1Department of Avian Science and Environmental Toxicology, University of California, Davis 95616, USA.
Abstract:
A method is presented for the isolation of a 155-kDa protein that possesses phenyl valerate hydrolysis activity in the presence of paraoxon but is inhibited by mipafox; the functional definition of neuropathy target esterase (neurotoxic esterase; NTE). Microsomes, isolated from 18-day-old chicken embryos were treated with phospholipase A2 to solubilize the NTE activity. The extract was then combined with polyoxyethylene W1 detergent and resolved by gel filtration chromatography to yield an active fraction with an approximate mass of 200 kDa. This fraction was further purified by preparative isoelectric focusing and native electrophoresis to yield two separate bands possessing NTE activity. The slower migrating band was highly enriched in a 155-kDa protein that was identified as a source of the NTE activity by affinity chromatography using 3-(9'-mercaptononylthio)-1,1,1-trifluoro-propan-2-one bound to Sepharose CL6B. This represents the first report of the isolation of NTE in its active form and aids in the confirmation of the 155-kDa protein as the most likely candidate for NTE.
Insights
Researchers isolated a 155-kDa protein with neuropathy target esterase (NTE) activity. This discovery confirms the 155-kDa protein as the likely source of NTE, advancing neurotoxic esterase research.
Area of Science:
- Biochemistry
- Neuroscience
- Enzymology
Background:
- Neuropathy target esterase (NTE), also known as neurotoxic esterase, is crucial for defining organophosphate-induced delayed neuropathy.
- Previous research has identified NTE activity but lacked a purified, active form of the enzyme for definitive characterization.
Purpose of the Study:
- To isolate and identify the active 155-kDa protein responsible for neuropathy target esterase (NTE) activity.
- To functionally characterize the isolated protein and confirm its role as NTE.
Main Methods:
- Solubilization of NTE activity from chicken embryo microsomes using phospholipase A2.
- Purification via gel filtration chromatography, preparative isoelectric focusing, and native electrophoresis.
- Affinity chromatography using a specific inhibitor to isolate the 155-kDa protein.
Main Results:
- Successful isolation of a 155-kDa protein exhibiting phenyl valerate hydrolysis activity, characteristic of NTE.
- The isolated 155-kDa protein was confirmed as the source of NTE activity through affinity chromatography.
- This marks the first report of NTE isolated in its active form.
Conclusions:
- The 155-kDa protein is confirmed as the most likely candidate for neuropathy target esterase (NTE).
- The successful isolation of active NTE provides a foundation for further biochemical and structural studies.
- This work enhances the understanding of NTE's role in neurotoxicity and esterase function.