Characterisation of the secretory acetylcholinesterases from adult Nippostrongylus brasiliensis.
The biochemical nature and relationship between the different isoforms of acetylcholinesterase (AChEs) secreted by adult Nippostrongylus brasiliensis was investigated, primarily via staining for enzyme activity and active-site labelling with [3H]-diisopropylfluorophosphate (DFP). Analysis by 1-dimensional SDS-PAGE under non-reducing conditions revealed the existence of 2 proteins of 74-kDa and 39-kDa, and each protein resolved as 2 species by isoelectric focusing. Both AChEs were co-purified via affinity chromatography on 9-[N beta-(epsilon-aminocaproyl)-beta-aminopropylamino]-acridine-coupled Sepharose 6B, and utilised to raise a polyclonal rabbit antiserum. Examination of the expression of secretory AChEs by adult worms during their residence in the gastrointestinal tract showed that the initial secretion of both forms on day 4 post-infection switched to predominant secretion of the 39-kDa protein by day 8. Immunoprecipitation of 35S-labelled products of in vitro translation via RNA from day 4 and day 8 worms predicted a single primary translation product of 59 kDa. These data suggested that the 'switching' event seen in vivo most likely corresponded to processing of the 74-kDa molecule. This interpretation was supported by limited digestion with V8 protease and chymotrypsin, which showed that the 74-kDa and the 39-kDa proteins possessed structural similarities.
Blackburn
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Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
Selkirk
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ME
D000110:Acetylcholinesterase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009559:Nippostrongylus / Q000201:enzymology* / Q000254:growth & development; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational
D011498:Protein Precursors; D000110:Acetylcholinesterase