IDENTIFICATION AND ISOLATION OF A 155-KDA PROTEIN WITH NEUROPATHY TARGET ESTERASE-ACTIVITY

A method is presented for the isolation of a 155-kDa protein that poss esses phenyl valerate hydrolysis activity in the presence of paraoxon but is inhibited by mipafox; the functional definition of neuropathy t arget esterase (neurotoxic esterase; NTE). Microsomes, isolated from 1 8-day-old chicken embryos were treated with phospholipase Az to solubi lize the NTE activity, The extract was then combined with polyoxyethyl ene W1 detergent and resolved by gel filtration chromatography to yiel d an active fraction with an approximate mass of 200 kDa, This fractio n was further purified by preparative isoelectric focusing and native electrophoresis to yield two separate bands possessing NTE activity, T he slower migrating band was highly enriched in a 155-kDa protein that was identified as a source of the NTE activity by affinity chromatogr aphy using (9'-mercaptononylthio)-1,1,1-trifluoropropan-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 pro tein as the most likely candidate for NTE. (C) 1996 Society of Toxicol ogy