Molecular characterisation of neuropathy target esterase: proteolysis of the [3H]DFP-labelled polypeptide.
Neuropathy target esterase (NTE) in hen brain membranes can be labelled with tritiated di-isopropylfluorophosphate ([3H]DFP) and appears to be associated with a 155-kDa polypeptide. Using preparative SDS-PAGE, we have obtained preparations in which [3H]DFP-labelled NTE comprises 2% of the total protein. Further purification of the 155-kDa polypeptide has proved difficult. We therefore attempted to use proteases to excise smaller [3H]DFP-labelled fragments which might be more amenable to fractionation. V8 protease treatment generated a labelled fragment of about 16 kDa which could be fractionated on SDS-PAGE and contained tritium attached to both site X (putatively the active site serine) and site Z (the residue to which an isopropyl moiety is transferred during aging of [3H]DFP-inhibited NTE). Papain and thermolysin treatments generated a small labelled peptide (< 10 kDa) which could be fractionated on reverse-phase HPLC and in which tritium was attached to site X but not site Z. N-terminal sequencing of the thermolysin-generated peptide fraction indicated sample heterogeneity but also suggested that the active site of NTE may contain the serine esterase consensus sequence: Gly-Glu-Ser-Xxx-Gly.
Glynn
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Medical Research Council Toxicology Unit, University of Leicester, UK.
Rüffer-Turner
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Read
D
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Wylie
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Johnson
M K
MK
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D005260:Female; D007531:Isoflurophate / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D014316:Tritium
D010446:Peptide Fragments; D014316:Tritium; D007531:Isoflurophate; D002265:Carboxylic Ester Hydrolases; C021859:neurotoxic esterase; D010447:Peptide Hydrolases