Nippostrongylus brasiliensis: characterisation of a somatic amphiphilic acetylcholinesterase with properties distinct from the secreted enzymes.
We have previously determined that Nippostrongylus brasiliensis secretes three monomeric nonamphiphilic (G1na) variants of acetylcholinesterase (AChE) with broadly similar properties. In this study we have examined AChE expression in somatic extracts of N. brasiliensis and report the identification of an additional enzyme which is not secreted. The enzyme was resolved by sucrose density gradient centrifugation with a sedimentation coefficient of 10.2 S which was shifted to 9.4 S in the presence of Triton X-100, identifying the enzyme as a tetrameric amphiphilic (G4a) form. The amphiphilic properties of this enzyme were confirmed by charge-shift electrophoresis, in which migration was accelerated by interaction with sodium deoxycholate. The enzyme showed low activity with butyrylthiocholine, and a Michaelis constant of 91 +/- 13 microM for acetylthiocholine was determined. It was highly sensitive to the AChE-specific inhibitor bis (4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with an IC50 of 6.5 +/- 0.4 microM, but was also inhibited by the butyrylcholinesterase-specific inhibitor tetramonoisopropylpyrophosphortetramide, albeit with a higher IC50 of 46.5 +/- 6.1 microM. This enzyme can therefore be distinguished from the secreted AChEs by its amphiphilic properties, sedimentation in sucrose gradients, and sensitivity to cholinesterase inhibitors.
Hussein
A S
AS
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, United Kingdom.
Grigg
M E
ME
Selkirk
M E
ME
D000109:Acetylcholine / Q000378:metabolism; D000110:Acetylcholinesterase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000122:Acetylthiocholine / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002093:Benzenaminium, 4,4'-(3-oxo-1,5-pentanediyl)bis(N,N-dimethyl-N-2-propenyl-), Dibromide / Q000494:pharmacology; D002092:Butyrylthiocholine / Q000037:antagonists & inhibitors / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D020128:Inhibitory Concentration 50; D009559:Nippostrongylus / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012044:Regression Analysis; D017206:Strongylida Infections / Q000469:parasitology; D013379:Substrate Specificity; D013770:Tetraisopropylpyrophosphamide / Q000494:pharmacology