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Necator americanus secretory acetylcholinesterase and its purification from excretory‐secretory products by affinity chromatography
Author(s) -
PRITCHARD D.I.,
LEGGETT K.V.,
ROGAN M.T.,
McKEAN P.G.,
BROWN ALAN
Publication year - 1991
Publication title -
parasite immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.795
H-Index - 75
eISSN - 1365-3024
pISSN - 0141-9838
DOI - 10.1111/j.1365-3024.1991.tb00274.x
Subject(s) - acetylcholinesterase , biology , necator americanus , aché , affinity chromatography , biochemistry , secretion , in vitro , antiserum , excretory system , enzyme , microbiology and biotechnology , antibody , immunology , endocrinology , helminths , ascaris lumbricoides
Summary Acetylcholinesterase (AChE) secretion by adult N. americanus was enhanced in vitro by incorporating insoluble collagen rafts into culture dishes. Enzyme produced in this way had preferential substrate specificity for acetylthio‐choline iodide (ATC), and its activity was inhibited by cscrine (1.1 × 10 −8 m ). Ancylostoma ceytanicum , another hookworm species, failed to produce comparable amounts of AChE in culture. AChE was efficiently purified from culture medium by affinity chromatography on edrophonium sepharose; 81% of the AChE activity was retained by the affinity matrix, although this fraction contained only 4.3% of the protein loaded. Antisera raised against purified AChE in rabbits immunohistochemically stained the oesophagcal glands of the parasite, and reacted with molecules of 32, 60, 80, 140 and 220 kDa in reduced adult ES products on Western blotting, although differential activity was observed against worm homogenatcs and earlier developmental stages. On IEF, purified AChE resolved predominately with a pl of 3.55; proteins with a similar pi were recognized by rabbit anti‐AChE. IgG preparations of this anliserum inhibited AChE activity in ES products, and inhibited AChE secretion by adult worms in culture. The availability of this immunological probe will allow definitive experiments to be conducted on the role of this enigmatic enzyme in the host‐parasite relationship.