AFFINITY-CHROMATOGRAPHY OF NEUROPATHY TARGET ESTERASE

Neuropathy target esterase (NTE) is a membrane-bound protein which has been proposed as the target site in nerve tissue for initiation of or ganophosphate induced delayed neuropathy (OPIDN). Efforts to character ize NTE and to determine the mechanism of its involvement in OPIDN hav e been hampered by the lack of a suitable method for its purification. We describe here the development of a trifluoromethyl ketone liganded affinity gel,which selectively binds NTE. Triton X-100/NaCl extracts of NTE from chick embryo brain microsomal membranes were adsorbed to a n affinity gel prepared by attachment of (9'-mercaptononylthio)-1,1,1- trifluoropropan-2-one to epoxy-activated Sepharose CL4B (MNTFP-Sepharo se). Typically 70 - 80% of NTE activity is bound under conditions in w hich undetectable quantities of total protein bound (< 4%). It proved difficult to elute active NTE under non-denaturing conditions, but SDS -PAGE analysis of MNTFP-Sepharose bound proteins eluted with 2% SDS id entified a 155 kDa NTE-like protein that bound in a trifluoromethylket one- or mipafox-sensitive but paraoxon-insensitive manner. The levels of inhibition of binding correlated with the inhibition of activity an d suggested that the 155-kDa band was composed of a single protein. MN TFP-Sepharose affinity chromatography in combination with preparative SDS-PAGE therefore holds promise as a method for obtaining microgram q uantities of NTE for chemical analysis and sequencing.