CHARACTERIZATION OF SALT-SOLUBLE FORMS OF ACETYLCHOLINESTERASE FROM BOVINE BRAIN

The hydrophilic, salt-soluble (SS) form of acetylcholinesterase (AChE) from bovine brain caudate nucleus exists mainly as a tetramer sedimen ting at 10.3S (similar to 40%), and a monomer sedimenting at 3.4S (sim ilar to 60%). The enzyme is N-glycosylated and contains similar HNK-1 carbohydrates as detergent-soluble (DS) AChE. No O-linked carbohydrate s could be detected. Amino acid sequencing showed that the N terminus of SS-AChE is identical to that of DS-AChE. In tetrameric SS-AChE, two pairs of disulfide-linked dimers are associated by hydrophobic forces located in the C terminus. Antibodies were raised against a peptide i dentical to the last 10 amino acid residues of bovine brain DS-AChE. T he peptide included the sequence of residues 574-583 (H-Tyr-Ser-Lys-Gl n-Asp-Arg-Cys-Ser-Asp-Leu-OH) of the enzyme. The antibodies cross-reac ted with tetrameric, but not with monomeric, SS-AChE, showing that in the latter form, the C terminus is truncated. Limited proteolysis of t etrameric SS-AChE at the C terminus led to the formation of an enzymat ically active monomer, which did not react with anti-C-terminal antibo dy. Although the DS form of AChE contains a structural subunit that se rves as membrane anchor, no anchor was detected in SS-AChE. Enzyme ant igen immunoassays showed that SS-AChE reacted with all monoclonal anti bodies directed against the catalytic subunit of DS-AChE, but not with monoclonal antibodies targeting the membrane-anchored subunits. From our results, we conclude that SS-AChE utilizes the same alternative sp licing pattern as DS-AChE, leading to tetrameric SS-AChE devoid of the membrane anchor. The active monomer of SS-AChE is most likely derived from tetrameric forms by limited postsynthetic proteolysis.