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Affinity chromatography of neuropathy target esterase.

Authors
Thomas-TC; Szekacs-A; Hammock-BD; Wilson-BW; McNamee-MG
Source
Chem-Biol Interact 1993 Jun; 87(1-3):347-360
NIOSHTIC No.
20032377
Abstract
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.
Keywords
Neuropathy; Nerve-tissue; Nerves; Chromatographic-analysis; Organo-phosphorus-compounds; Ketones; Chemical-analysis; Molecular-structure; Metabolic-study; Filtration
Contact
Thomas C. Thomas, Brigham and Women's Hospital, Thorn Building, Room 1123, 75 Francis St., , Boston, MA 02115, USA.
CODEN
CBINA8
Publication Date
19930601
Document Type
Journal Article
Funding Amount
1055222
Funding Type
Cooperative Agreement
Fiscal Year
1993
NTIS Accession No.
NTIS Price
Identifying No.
Cooperative-Agreement-Number-U07-CCU-906162
Issue of Publication
1-3
ISSN
0009-2797
Source Name
Chemico-Biological Interactions
State
CA
Performing Organization
University of California - Davis
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