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 organophosphate induced delayed neuropathy (OPIDN). Efforts to characterize NTE and to determine the mechanism of its involvement in OPIDN have 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 an affinity gel prepared by attachment of 3(9'-mercaptononylthio)-1,1,1-trifluoropropan-2-one to epoxy-activated Sepharose CL4B (MNTFP-Sepharose). Typically 70-80% of NTE activity is bound under conditions in which 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 identified a 155 kDa NTE-like protein that bound in a trifluoromethylketone- or mipafox-sensitive but paraoxon-insensitive manner. The levels of inhibition of binding correlated with the inhibition of activity and suggested that the 155-kDa band was composed of a single protein. MNTFP-Sepharose affinity chromatography in combination with preparative SDS-PAGE therefore holds promise as a method for obtaining microgram quantities of NTE for chemical analysis and sequencing.
Thomas
T C
TC
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Székács
A
A
Hammock
B D
BD
Wilson
B W
BW
McNamee
M G
MG
D000096:Acetone / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D002642:Chick Embryo; D002846:Chromatography, Affinity; D007700:Kinetics; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000138:chemical synthesis / Q000378:metabolism
D013438:Sulfhydryl Compounds; D000096:Acetone; C082482:3-(9'-mercaptononylthio)-1,1,1,-trifluoropropan-2-one; D012685:Sepharose; D002265:Carboxylic Ester Hydrolases; C021859:neurotoxic esterase