Dimeric forms of cholinesterase in Sipunculus nudus.
In developing a research on the cholinesterase (ChE) evolution in Invertebrata, this enzyme was studied in the unsegmented marine worm Sipunculus nudus. ChE activity was solubilized through three successive steps of extraction. These fractions are noted as low-salt (LSS), detergent (DS) and high-salt soluble (HSS) and represent 27%, 68% and 5% of total activity, respectively. LSS and DS ChE were purified to homogeneity by affinity chromatography on edrophonium-Sepharose gel. Purification factors of 1700 (LSS) and 1090 (DS) were obtained. The small amount of HSS ChE prevented a similar purification and an extensive characterization. Based on SDS/PAGE and density-gradient centrifugation, both LSS and DS enzymes show a M(r) value of about 130,000 and are likely G2 globular dimers of a 67,000 subunit. Moreover, LSS ChE seems to be an amphiphilic form including a hydrophobic domain, while DS ChE is probably 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 other thiocholine esters as substrates, thus suggesting a wide and little-specialized conformation of the active site. Based on immunological cross-reactivity trials, LSS and DS ChE from S. nudus show a reduced structural affinity with a molluscan (Murex brandaris) enzyme. HSS ChE, an acetylcholinesterase, is also solubilized by heparin, like typical vertebrate HSS asymmetric enzymes. However, it lacks fast-sedimenting forms and an enzyme-anchoring collagenous structure.
Talesa
V
V
Department of Experimental Medicine, University of Perugia, Italy.
Principato
G B
GB
Giovannini
E
E
Di Giovanni
M V
MV
Rosi
G
G
D000818:Animals; D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D002802:Cholinesterases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D017364:Collagenases / Q000378:metabolism; D003429:Cross Reactions; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000378:metabolism; D015151:Immunoblotting; D008970:Molecular Weight; D009348:Nematoda / Q000201:enzymology*; D011487:Protein Conformation; D012995:Solubility; D010738:Type C Phospholipases / Q000378:metabolism
D003902:Detergents; D006493:Heparin; D002802:Cholinesterases; D010738:Type C Phospholipases; D017364:Collagenases