ACETYLCHOLINESTERASE IN SPIROGRAPHIS-SPALLANZANII (POLYCHAETA, SEDENTARIA) - PRESENCE OF 2 DIMERIC MEMBRANE-BOUND FORMS

In the annelid polychaete Spirographis spallanzanii two acetylcholines terases, named DS and HSDS, were detected. They differ in relative amo unt, membrane anchoring and pharmacological properties. Studies with i nhibitors evidenced complete inhibition of both acetylcholinesterases by 10(-3) M eserine and different sensitivities for edrophonium or pro cainamide. Both enzymes, sensitive to BW284c51, were unaffected by iso -OMPA; at variance, only the HSDS form underwent excess-substrate inhi bition. DS and HSDS enzymes were solubilized by homogenization In a lo w-salt or high-salt-Triton X-100 buffer and then purified by affinity chromatography on edrophonium or procainamide-Sepharose column respect ively. According to gel-filtration chromatography, sedimentation analy sis and SDS-PAGE, the least represented (30%) DS form is a G(2) amphip hilic globular dimer (124-130 kDa, 6.0-7.0S) with S-S linked monomers (66 kDa). Phosphatidylinositol anchors give cell membrane insertion, s elf-aggregation and detergent (Triton X-100, Brij 97) interaction. The prevailing (70%) HSDS acetylcholinesterase is once again a G(2) form similar to DS enzyme in its molecular size (117-125 kDa), sedimentatio n coefficient (6.0S) of the native form and presence of S-S linked sub units (66 kDa). However, it is likely attached to the cell membrane by involvement of strong electrostatic interactions. DS acetylcholineste rase displays moderate active site specificity with differently sized substrates. The HSDS form is inactive on butyrylthiocholine. DS and HS DS forms show a comparable catalytic efficiency (k(cat)/K-m) approachi ng that of other invertebrate enzymes. The results suggest that DS and HSDS enzymes, likely encoded by distinct genes, are both functional i n cholinergic synapses.