Monomerization of tetrameric bovine caudate nucleus acetylcholinesterase. Implications for hydrophobic assembly and membrane anchor attachment site.
Tetrameric detergent-soluble bovine caudate nucleus acetylcholinesterase (AChE) was reduced and alkylated under conditions in which at least 95% of initial activity is retained. This treatment alone did not result in monomerization of AChE, nor did it create a hydrophilic enzyme. However, in the presence of SDS the enzyme became monomerized. Incubation of AChE with trypsin in the presence of the reversible inhibitor edrophonium rendered the enzyme hydrophilic and led to catalytically active monomers being produced. SDS/PAGE of this preparation in non-reducing conditions revealed only a small decrease in the subunit molecular mass. N-Terminal sequencing of the enzyme, before and after trypsin treatment, yielded identical N-termini showing that the enzyme was monomerized subsequent to C-terminal tryptic cleavage. From our results, we conclude that the most C-terminal cysteine residue is involved in inter-subunit disulphide bonding as well as in the attachment of AChE to the membrane anchor. Furthermore, the C-terminal region in the primary structure provides an area for hydrophobic contacts between the different subunits and also between the subunits and the membrane anchor.
Heider
H
H
Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Brodbeck
U
U
D000110:Acetylcholinesterase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002421:Caudate Nucleus / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008961:Models, Structural; D008970:Molecular Weight; D017830:Octoxynol; D010446:Peptide Fragments / Q000302:isolation & purification; D011092:Polyethylene Glycols; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D003902:Detergents; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D017830:Octoxynol; D000110:Acetylcholinesterase