Acetylcholinesterase in Spirographis spallanzanii (Polychaeta: Sedentaria): presence of two dimeric membrane-bound forms.
In the annelid polychaete Spirographis spallanzanii two acetylcholinesterases, named DS and HSDS, were detected. They differ in relative amount, membrane anchoring and pharmacological properties. Studies with inhibitors evidenced complete inhibition of both acetylcholinesterases by 10(-3) M eserine and different sensitivities for edrophonium or procainamide. Both enzymes, sensitive to BW284c51, were unaffected by iso-OMPA; at variance, only the HSDS form underwent excess-substrate inhibition. DS and HSDS enzymes were solubilized by homogenization in a low-salt or high-salt-Triton X-100 buffer and then purified by affinity chromatography on edrophonium- or procainamide-Sepharose column respectively. According to gel-filtration chromatography, sedimentation analysis and SDS-PAGE, the least represented (30%) DS form is a G2 amphiphilic globular dimer (124-130 kDa, 6.0-7.0S) with S-S linked monomers (66 kDa). Phosphatidylinositol anchors give cell membrane insertion, self-aggregation and detergent (Triton X-100, Brij 97) interaction. The prevailing (70%) HSDS acetylcholinesterase is once again a G2 form similar to DS enzyme in its molecular size (117-125 kDa), sedimentation coefficient (6.0S) of the native form and presence of S-S linked subunits (66 kDa). However, it is likely attached to the cell membrane by involvement of strong electrostatic interactions. DS acetylcholinesterase displays moderate active site specificity with differently sized substrates. The HSDS form is inactive on butyrylthiocholine. DS and HSDS forms show a comparable catalytic efficiency (kcat/K(m)) approaching that of other invertebrate enzymes. The results suggest that DS and HSDS enzymes, likely encoded by distinct genes, are both functional in cholinergic synapses.
Talesa
V
V
Department of Experimental Medicine, University of Perugia, Italy.
Romani
R
R
Rosi
G
G
Giovannini
E
E
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D011077:Polychaeta / Q000201:enzymology*; D011487:Protein Conformation; D010738:Type C Phospholipases
D007527:Isoenzymes; D008565:Membrane Proteins; D000110:Acetylcholinesterase; D010738:Type C Phospholipases