SOLUBILIZATION, MOLECULAR-FORMS, PURIFICATION AND SUBSTRATE-SPECIFICITY OF 2 ACETYLCHOLINESTERASES IN THE MEDICINAL LEECH (HIRUDO-MEDICINALIS)

Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo med icinalis). A 'spontaneously-soluble' portion of AChE activity (SS-AChE ) was recovered from haemolymph and from tissues dilacerated in low-sa lt buffer. A second portion of AChE activity was obtained after extrac tion of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogen eity by affinity chromatography on edrophonium- and concanavalin A-Sep harose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native S S-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedime nting at 5.0 S in sucrose gradients with or without Triton X-100, sugg esting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-spe cific phospholipase C suppressed aggregation and gave a 7 S peak. DS-A ChE was thus an amphiphilic glycolipid-anchored dimer. Substrate speci ficities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-A ChE displayed only limited variations in K-m values with charged and u ncharged substrates, suggesting a reduced influence of electrostatic i nteractions in the enzyme substrate affinity. By contrast, DS-AChE dis played higher K-m values with uncharged than with charged substrates, SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M).