Nippostrongylus brasiliensis: Characterisation of a somatic amphiphilic acetylcholinesterase with properties distinct from the secreted enzymes

We have previously determined that Nippostrongylus brasiliensis secretes th ree monomeric nonamphiphilic (G(1)(na)) variants of acetylcholinesterase (A ChE) with broadly similar properties. In this study we have examined AChE e xpression in somatic extracts of N, brasiliensis and report the identificat ion 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, iden tifying the enzyme as a tetrameric amphiphilic (G(4)(a)) form. The amphiphi lic properties of this enzyme were confirmed by charge-shift electrophoresi s, in which migration was accelerated by interaction with sodium deoxychola te. The enzyme showed low activity with butyrylthiocholine, and a Michaelis constant of 91 +/- 13 mu M for acetylthiocholine was determined. It was hi ghly sensitive to the AChE- specific inhibitor bis (4-allyldimethylammonium phenyl)pentan-3-one dibromide, with an IC50 of 6.5 +/- 0.4 mu M. but was al so inhibited by the butyrylcholinesterase- specific inhibitor tetramonoisop ropylpyrophosphortetramide. albeit with a higher IC50 of 96.5 +/- 6.1 mu M This enzyme can therefore be distinguished from the secreted AChEs by its a mphiphilic properties, sedimentation in sucrose gradients. and sensitivity to cholinesterase inhibitors. (C) 1999 Academic Press.