Biochemical and molecular characterization of acetylcholinesterase from the hagfish Myxine glutinosa.
To obtain information about the evolution of the cholinesterases, we investigated the cholinesterase activity of an agnathan vertebrate, the hagfish Myxine glutinosa. On the basis of evidence from enzymology, pharmacology, and molecular biology, we conclude that the cholinesterase activity is due to acetylcholinesterase (AChE). The enzyme hydrolyzes acetylthiocholine preferentially and exhibits substrate inhibition. The hydrolysis of both acetylthiocholine and butyrylthiocholine are inhibited in parallel by cholinesterase inhibitors, with the AChE-specific drug BW284c51 being the most potent; however, this drug and propidium, a peripheral anionic site ligand, are much weaker inhibitors of the hagfish enzyme than of Torpedo AChE. We used sequential extraction, collagenase digestion, and velocity sedimentation on sucrose gradients to determine that the AChE from the skeletal muscle of the hagfish is present in both globular and asymmetric forms. We also used the polymerase chain reaction with degenerate oligonucleotide probes and genomic DNA to obtain a 1 kb gene fragment for hagfish AChE. The enzyme has an acyl binding site typical of other vertebrate AChE, but lacks two aromatic residues implicated in the function of the peripheral anionic subsite. We discuss the relevance of our findings to the evolution of the cholinesterases in the vertebrates.
Division of Science and Mathematics, Birmingham-Southern College, AL 35254, USA.
M
M
Sanders
B
B
Mathews
D
D
Sutherland
W
W
Soong
H
H
Giles
L
L
Pezzementi
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017364:Collagenases / Q000378:metabolism; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry; D006196:Hagfishes; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment
D004247:DNA; D000110:Acetylcholinesterase; D017364:Collagenases