2 CHOLINESTERASE ACTIVITIES AND GENES ARE PRESENT IN AMPHIOXUS

To obtain information about the evolution of the cholinesterases, acet ylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the verte brates, we investigated the cholinesterase (ChE) activity of the cepha lochordate amphioxus (Branchiostoma floridae and Branchiostoma lanceol atum). On the basis of evidence from enzymology, pharmacology, and mol ecular biology, we conclude that amphioxus possesses two ChE activitie s and two ChE genes. Two covalent inhibitors of cholinesterases were a ble to pharmacologically isolate the two activities as drug-sensitive ChE: and drug-resistant ChE. Kinetically, in terms of substrate specif icity, the drug-sensitive ChE resembles vertebrate AChE, and the drug- resistant ChE resembles the BuChE of cartilaginous and bony fish or th e intermediate ChE of protostome invertebrates. We also used the polym erase chain reaction with degenerate oligonucleotide primers and genom ic DNA to obtain clones of 1,574 and 1,011 bp corresponding to two cho linesterase genes from amphioxus, which we designated as ChE1 and ChE2 . ChE2 codes for an enzyme with an acyl-binding pocket sequence, a por tion of the protein that plays an important role in determining substr ate specificity, typical of invertebrate ChE. ChE1, which contains a 5 03-bp intron, encodes a protein with a novel, acyl binding site. Phylo genetic analysis of the sequences suggests that the two genes are a re sult of a duplication event in the lineage leading to amphioxus. We di scuss the relevance of our results to the evolution of the cholinester ases in the chordates. Previously, we reported that amphioxus containe d a single cholinesterase activity with properties intermediate to ACh E: and BuChE (Pezzementi et al. [1991] In: Cholinesterases: Structure, Function, Mechanism, Genetics and Cell Biology. J. Massoulie et al., eds. ACS: Washington, D.C., pp. 24-31). (C) 1997 Wiley-Liss, Inc.