DIMERIC FORMS OF CHOLINESTERASE IN SIPUNCULUS-NUDUS

In developing a research on the cholinesterase (ChE) evolution in Inve rtebrata, this enzyme was studied in the unsegmented marine worm Sipun culus nudus. ChE activity was solubilized through three successive ste ps of extraction. These fractions are noted as low-salt (LSS), deterge nt (DS) and high-salt soluble (HSS) and represent 27%, 68% and 5% of t otal activity, respectively. LSS and DS ChE were purified to homogenei ty by affinity chromatography on edrophonium-Sepharose gel. Purificati on factors of 1700 (LSS) and 1090 (DS) were obtained. The small amount of HSS ChE prevented a similar purification and an extensive characte rization. Based on SDS/PAGE and density-gradient centrifugation, both LSS and DS enzymes show a M(r) value of about 130000 and are likely G2 globular dimers of a 67000 subunit. Moreover, LSS ChE seems to be an amphiphilic form including a hydrophobic domain, while DS ChE is proba bly linked to the cell membrane by a phosphatidylinositol anchor. Both LSS and DS enzymes hydrolyze at the highest rate propionylthiocholine . However, they also show a fairly high catalytic efficiency with othe r thiocholine esters as substrates, thus suggesting a wide and little- specialized conformation of the active site. Based on immunological cr oss-reactivity trials, LSS and DS ChE from S. nudus show a reduced str uctural affinity with a molluscan (Murex brandaris) enzyme. HSS ChE, a n acetylcholinesterase, is also solubilized by heparin, like typical v ertebrate HSS asymmetric enzymes. However, it lacks fast-sedimenting f orms and an enzyme-anchoring collagenous structure.