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.
| Original language | English (US) |
|---|---|
| Article number | 0039 |
| Pages (from-to) | 23-30 |
| Number of pages | 8 |
| Journal | Toxicological Sciences |
| Volume | 30 |
| Issue number | 1 |
| DOIs | |
| State | Published - 1996 |
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ASJC Scopus subject areas
- Toxicology
Cite this
Identification and isolation of a 155-kDa protein with neuropathy target esterase activity. / Mackay, Christopher E.; Hammock, Bruce D.; Wilson, Barry W.
In: Toxicological Sciences, Vol. 30, No. 1, 0039, 1996, p. 23-30.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Identification and isolation of a 155-kDa protein with neuropathy target esterase activity
AU - Mackay, Christopher E.
AU - Hammock, Bruce D.
AU - Wilson, Barry W.
PY - 1996
Y1 - 1996
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85033041897&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85033041897&partnerID=8YFLogxK
U2 - 10.1093/toxsci/30.1.23
DO - 10.1093/toxsci/30.1.23
M3 - Article
AN - SCOPUS:85033041897
VL - 30
SP - 23
EP - 30
JO - Toxicological Sciences
JF - Toxicological Sciences
SN - 1096-6080
IS - 1
M1 - 0039
ER -