Acetylcholinesterase in Dendrobaena veneta (Oligochaeta: Opisthopora) is Present with Forms Sensitive and Insensitive to Phosphatidylinositol Phospholipase C

Abstract

Three distinct acetylcholinesterases were detected in the annelid oligochaete Dendrobuenu veneta.Two enzymes (a, p), copurified from a Triton-X-l 00-soluble extract of whole animals by affinity (edrophonium-Sepharose) chromatography, were separately eluted from a Sephadex G-200 column. Gel-filtration chromatography, sedimentation analysis and SDS/PAGE showed the a and p forms to be a globular dimer (1 10 kDa, 7.0 S) and a hydrophilic monomer (58 kDa, 5.0 S) respectively, both weakly linked to the cell membrane. The third form ( y ) , also purified to homogeneity by slower filtration through an edrophonium-Sepharose matrix, proved to be an amphiphilic globular dimer ( 1 33 kDa, 7.0 S) with a phosphatidylinositol anchor giving cell membrane insertion, detergent (Triton X-100, Brij 96) interaction and self-aggregation. The a acetylcholinesterase showed a fairly low substrate specificity : the p form hydrolyzed propionylthiocholine at the highest rate and was inactive on butyrylthiocholine ; the y acetylcholinesterase, showing a marked active-site specificity with differently sized substrates, was likely functional in cholinergic synapses. Studies with inhibitors showed incomplete inhibition of all three acetylcholinesterase by 1 mM eserine and different sensitivity for edrophonium or procainamide. The a and p forms, sensitive to 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, were unaffected by tetra(monoisopropy1)-pyrophosphortetramide, while both these agents inhibited the y enzyme. All three forms showed excess-substrate inhibition by acetylthiocholine. Enzyme activity was histochemically localized in the nerve ring and its minor branches. Monomeric acetylcholinesterase (p) is likely the only form present in the ganglionic glial framework.

Effects of chlorpyrifos on the catalytic efficiency and expression level of acetylcholinesterases in the bivalve mollusk Scapharca inaequivalvis

“…Density gradient centrifugation was performed as previously described [6,14] to assess the possible changes in the expression of AChE molecular forms due to CPF treatments. In particular, samples (250 l) of LST or LS extract from either foot or gills, as well as of purified fractions after affinity chromatography, were layered onto 5 to 20% sucrose density gradients (10 ml in polyallomer tubes) in high-salt or high-salt-Triton buffer.…”

“…Such enzymes may exist in multiple molecular forms as monomers or oligomers of either hydrophilic or amphiphilic catalytic subunits both in Vertebrata [1] and Invertebrata [2][3][4][5][6]. Such enzymes may exist in multiple molecular forms as monomers or oligomers of either hydrophilic or amphiphilic catalytic subunits both in Vertebrata [1] and Invertebrata [2][3][4][5][6].…”

Molecular Cloning and Expression of a Full‐Length cDNA Encoding Acetylcholinesterase in Optic Lobes of the Squid

“…Density gradient centrifugation to assess hydrodynamic properties of the enzyme, SDS‐polyacrylamide gel electrophoresis to study quaternary structure and molecular weight of purified enzyme, treatment of AChE with phospholipase C (Boehringer Mannheim), and assay procedures were performed as previously described (Talesa et al, 1996). In particular, purified AChE from optic lobe was always used at a final concentration of 2 n M .…”

“…In particular, purified AChE from optic lobe was always used at a final concentration of 2 n M . Propidium (Sigma), a reversible inhibitor of the AChE peripheral site, and diisopropyl fluorophosphate (DFP ; Sigma), an irreversible inhibitor of the AChE active site, were also used in inhibition studies as previously described (Arpagaus et al, 1994 ; Talesa et al, 1996).…”

“…Only globular forms of AChE are present in invertebrates (Talesa et al, 1996). A dimeric AChE linked to the cell membrane by a phosphatidylinositol (PtdIns) anchor is always expressed in the organisms so far studied, alone or together with other globular forms, as, for instance, in Musca domestica (Fournier et al, 1988) and in Drosophila (Toutant et al, 1988).…”