Molecular Forms of Acetylcholinesterase from Human Caudate Nucleus: Comparison of Salt‐Soluble and Detergent‐Soluble Tetrameric Enzyme Species

Abstract

Extraction of human caudate nucleus under high-ionic-strength conditions solubilized 20-30% of total acetylcholinesterase (AChE) activity. Density gradient centrifugation revealed monomeric (5.0 S) and tetrameric (11.0 S) enzyme species. The purified, tetrameric salt-soluble (SS) AChE sedimented at 10.6 S and did not bind detergents. It showed an immunochemical reaction of identity with the detergent-soluble (DS) AChE species from human caudate nucleus and human erythrocytes, but did not cross-react with antibodies raised against human serum cholinesterase. The remaining activity was solubilized under low-ionic-strength conditions in the presence of 1.0% Triton X-100. The purified tetrameric, DS-AChE sedimented at 10.0 S as detergent-protein mixed micelle and on extensive removal of the detergent this enzyme formed defined aggregates by self-micellarization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed that the salt-soluble and detergent-soluble tetrameric enzyme species both contained a heavy and a light dimer; under reducing conditions mainly one band corresponding to the light subunit was seen. Molecular weights of 300,000 dalton and 280,000 dalton were calculated for SS-AChE and DS-AChE, respectively. Limited digestion of DS-AChE with proteinase K led to isolation of an enzyme that no longer bound detergents and lacked the intersubunit disulfide bridges.

The membrane anchor of mammalian brain acetylcholinesterase consists of a single glycosylated protein of 22 kDa

“…Detergent-soluble tetrameric AChE containing the P-subunit was purified from frozen calf brain essentially as described by Gennari and Brodbeck [11] using an affinity resin with trimethyl-ammonium mphenylenediamine as affinity ligand [12]. All steps were performed at 4°C in presence of 20 mM EDTA and 2 mM benzamidine hydrochloride as protease inhibitors.…”

Acetylcholinesterases of the nematode Steinernema carpocapsae

“…7). This situation is at variance to that of G4 forms of vertebrate cholinesterases where such dissociation of tetramers is usually not achieved by reducing agents [31, 321 or removal of S-S bonds by limited proteolyis [33,341. The forces holding the subunits together in the G4 form of AChE were recently shown to result from a hydrophobic sequence located near the Cterminus of the catalytic peptide [35].…”

Association of Acetylcholinesterase with the cell surface

“…Previous studies have shown that the hydrophobic G4 AChE form could be converted to a catalytically active hydrophilic enzyme after protease treatment (Gennari & Brodbeck, 1985). We submitted the ~25I-TID-labeled AChE to the action of proteinase K. After this treatment 80% of the activity remained; however, almost all the radioactivity associated with the G 4 AChE was released, and instead of the 20-kDa labeled band most of the label migrated close to the front of the SDS-polyacrylamide gel, with an approximate molecular mass of 13 kDa, indicating that proteinase K released most of the hydrophobic domain from the enzyme (Fuentes .…”