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 sedimenting at 10.3S (approximately 40%), and a monomer sedimenting at 3.4S (approximately 60%). The enzyme is N-glycosylated and contains similar HNK-1 carbohydrates as detergent-soluble (DS) AChE. No O-linked carbohydrates 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 identical to the last 10 amino acid residues of bovine brain DS-AChE. The peptide included the sequence of residues 574-583 (H-Tyr-Ser-Lys-Gln-Asp-Arg-Cys-Ser- Asp-Leu-OH) of the enzyme. The antibodies cross-reacted with tetrameric, but not with monomeric, SS-AChE, showing that in the latter form, the C terminus is truncated. Limited proteolysis of tetrameric SS-AChE at the C terminus led to the formation of an enzymatically active monomer, which did not react with anti-C-terminal antibody. Although the DS form of AChE contains a structural subunit that serves as membrane anchor, no anchor was detected in SS-AChE. Enzyme antigen immunoassays showed that SS-AChE reacted with all monoclonal antibodies 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 splicing pattern as DS-AChE, leading to tetrameric SS-AChE devoid of the membrane anchor.(ABSTRACT TRUNCATED AT 250 WORDS)
Liao
J
J
Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland.
Boschetti
N
N
Mortensen
V
V
Jensen
S P
SP
Koch
C
C
Nørgaard-Pedersen
B
B
Brodbeck
U
U
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001921:Brain / Q000201:enzymology*; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002421:Caudate Nucleus / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D003429:Cross Reactions; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology; D012492:Salts; D012995:Solubility
D000906:Antibodies; D002241:Carbohydrates; D004220:Disulfides; D007527:Isoenzymes; D046911:Macromolecular Substances; D009842:Oligopeptides; D012492:Salts; D000110:Acetylcholinesterase