title: Characterisation of the secretory acetylcholinesterases from adult Nippostrongylus brasiliensis.
abstract: 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.
authors.affiliation: Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
authors.forename: C C
authors.initials: CC
authors.lastname: Blackburn
authors.forename: M E
authors.initials: ME
authors.lastname: Selkirk
mesh_terms: 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
chemical_list: D011498:Protein Precursors; D000110:Acetylcholinesterase