The molecular forms of acetylcholinesterase from Necator americanus (Nematoda), a hookworm parasite of the human intestine.
Necator americanus (Nematoda: Strongyloidea), a human hookworm parasite, is known to release considerable amounts of acetylcholinesterase (AChE) [Pritchard, D. I., Leggett K. V., Rogan, M. T., McKean, P. G. & Brown, A. (1991) Necator americanus secretory acetylcholinesterase and its purification from excretory/secretory products by affinity chromatography, Parasite Immunol. 13, 187-199]. The present study deals with AChE activity recovered in sequential somatic extracts, and excretory/secretory products, of the adult stage of the parasite. 97% of AChE was extractable in low-salt and high-salt detergent-free buffers, and only 3% was solubilised by a further extraction in the presence of Triton X-100. AChE in all three extracts was affected by the AChE inhibitors eserine, bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide and edrophonium chloride, but was resistant to the effects of tetramonoisopropylpyrophosphortetramide, a butyrylcholinesterase inhibitor. Sucrose density centrifugation revealed that AChE in all somatic extracts (low-salt, high-salt and detergent) resolved almost exclusively as a single peak between 6.9-7.5 S, while excretory/secretory products resolved at 8.2 S. These values are all compatible with dimers of catalytic subunits and no evidence was found for the presence of higher oligomers such as asymmetric forms. The only sample to show a shift in sedimentation following the inclusion of detergent (Triton X-100, Brij 96) in the gradient was a component of the detergent-soluble extract, indicating the existence of a minor amphiphilic form. In low-salt-soluble and high-salt-soluble extracts, AChE was solubilised as a hydrophilic globular form, probably a dimeric G2. The analysis of diisopropylfluorophosphate-labelled extracts by SDS/PAGE, and unlabelled extracts by immunoblotting using a polyvalent antiserum to N. americanus AChE, indicated that the AChE isolated in each extract was biochemically and immunologically similar. The banding patterns obtained were comparable to that seen when purified AChE was analysed by SDS/PAGE and immunoblotted. This suggests that the basic catalytic subunit has a mass of 66-70 kDa with the active site being located in a 30-kDa domain. All experimental data indicate the existence of only one AChE class in Necator homologous to AChE of class B from Caenorhabditis elegans. The solubility characteristics and globular nature of this hookworm AChE suggest that its major function is as an excretory or secretory product. This again raises the question of the true biological function of this 'non-cholinergenic' nematode secretion.
Pritchard
D I
DI
Department of Life Science, University of Nottingham, England.
Brown
A
A
Toutant
J P
JP
D000110:Acetylcholinesterase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007531:Isoflurophate / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D017221:Necator americanus / Q000201:enzymology* / Q000472:pathogenicity; D009332:Necatoriasis / Q000469:parasitology*; D013379:Substrate Specificity
D007527:Isoenzymes; D007531:Isoflurophate; D000110:Acetylcholinesterase