Molecular cloning and expression of a full-length cDNA encoding acetylcholinesterase in optic lobes of the squid Loligo opalescens: a new member of the cholinesterase family resistant to diisopropyl fluorophosphate.
Acetylcholinesterase cDNA was cloned by screening a library from Loligo opalescens optic lobes; cDNA sequence analysis revealed an open reading frame coding for a protein of 610 amino acids that showed 20-41% amino acid identity with the acetylcholinesterases studied so far. The characteristic structure of cholinesterase (the choline binding site, the catalytic triad, and six cysteines that form three intrachain disulfide bonds) was conserved in the protein. The heterologous expression of acetylcholinesterase in COS cells gave a recovery of acetylcholinesterase activity 20-fold higher than in controls. The enzyme, partially purified by affinity chromatography, showed molecular and kinetic features indistinguishable from those of acetylcholinesterase expressed in vivo, which displays a high catalytic efficiency. Both enzymes are true acetylcholinesterase corresponding to phosphatidylinositol-anchored G2a dimers of class I, with a marked substrate specificity for acetylthiocholine. The deduced amino acid sequence may explain some particular kinetic characteristics of Loligo acetylcholinesterase, because the presence of a polar amino acid residue (S313) instead of a nonpolar one [F(288) in Torpedo] in the acyl pocket of the active site could justify the high substrate specificity of the enzyme, the absence of hydrolysis with butyrylthiocholine, and the poor inhibition by the organophosphate diisopropyl fluorophosphate.
Department of Experimental Medicine, University of Perugia, Italy.
V
V
Talesa
M
M
Grauso
M
M
Arpagaus
E
E
Giovannini
R
R
Romani
G
G
Rosi
D000110:Acetylcholinesterase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000187:drug effects* / Q000201:enzymology; D019556:COS Cells; D002499:Centrifugation, Density Gradient; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D049832:Decapodiformes / Q000378:metabolism*; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D007531:Isoflurophate / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D010738:Type C Phospholipases / Q000737:chemistry
D002800:Cholinesterase Inhibitors; D018076:DNA, Complementary; D007531:Isoflurophate; D000110:Acetylcholinesterase; D010738:Type C Phospholipases