Identification of a trypsin-like site associated with acetylcholinesterase by affinity labelling with [3H]diisopropyl fluorophosphate.
In addition to its ability to hydrolyze acetylcholine, purified eel acetylcholinesterase possesses a trypsin-like endopeptidase activity. The tryptic activity is associated with a serine residue at a site that is distinct from the esteratic site. To label both the esteratic and tryptic sites, the enzyme was incubated with the serine hydrolase inhibitor [3H]diisopropyl fluorophosphate. This compound labelled the protein in a biphasic manner, with both slow and rapid labelling kinetics. The time course of the rapid phase was similar to the time course of inactivation of the esteratic activity. The time course of the slow phase was similar to the time course of inactivation of the tryptic activity. Labelling of the nonesteratic site was inhibited by the trypsin inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone. The total number of sites labelled by [3H]diisopropyl fluorophosphate on eel acetylcholinesterase was 2.6 mol/280,000 g protein, whereas the number of tryptic sites was less (0.52 mol/280,000 g). The results suggest that a subpopulation of acetylcholinesterase molecules may possess tryptic activity. Extensive chromatography of the purified enzyme by ion-exchange and gel filtration failed to separate the labelled tryptic component from acetylcholinesterase. On sodium dodecyl sulfate-polyacrylamide gels, the labelled tryptic component comigrated with a polypeptide of 50,000 molecular weight, which is a major proteolytic digestion product derived from the intact acetylcholinesterase monomer. Because of its localization in many noncholinergic peptide-containing cells, acetylcholinesterase could act as a neuropeptide processing enzyme in these cells.
Small
D H
DH
Department of Biochemistry, University of Melbourne, Parkville, Victoria.
Chubb
I W
IW
D000110:Acetylcholinesterase / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002845:Chromatography; D004524:Eels; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000378:metabolism; D007531:Isoflurophate / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010830:Physostigmine / Q000494:pharmacology; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014316:Tritium; D014357:Trypsin / Q000378:metabolism*
D000345:Affinity Labels; D010446:Peptide Fragments; D014316:Tritium; D007531:Isoflurophate; D014107:Tosyllysine Chloromethyl Ketone; D010830:Physostigmine; D004950:Esterases; D000110:Acetylcholinesterase; D014357:Trypsin