Bovine Nucleus Caudatus Acetylcholinesterase: Active Site Determination and Investigation of a Dimeric Form Obtained by Selective Proteolysis

The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.

Section: Discussionsupporting

confidence: 89%

Acetylcholinesterase in Mouse Neuroblastoma NB₂A Cells: Analysis of Production, Secretion, and Molecular Forms

Stieger

Bütikofer

Wiesmann

et al. 1989

“…It is possible that such forms were, in fact, aggregates of amphiphilic dimers which, once established, resisted dissociation by detergents. Such a phenomenon was reported recently by Landauer et al (1984), who observed that a G4 form from bovine caudate nucleus AChE produced welldefined 16s and 20s aggregates. In our hands, the HSS enzyme of Drosophila was composed only of dimers similar to those recovered in the LSS and DS fractions; we thus concluded that asymmetric forms do not exist in the case of Drosophila AChE.…”

Section: Discussionsupporting

confidence: 77%

Native Molecular Forms of Head Acetylcholinesterase from Adult Drosophila melanogaster: Quaternary Structure and Hydrophobic Character

Toutant¹,

Arpagaus

Fournier

1988

The native molecular forms of acetylcholinesterase (AChE) present in adult Drosophila heads were characterized by sedimentation analysis in sucrose gradients and by nondenaturing electrophoresis. The hydrophobic properties of AChE forms were studied by comparing their migration in the presence of Triton X100, 10-oleyl ether, or sodium deoxycholate, or in the absence of detergent. We examined the polymeric structure of AChE forms by disulfide bridge reduction. We found that the major native molecular form is an amphiphilic dimer which is converted into hydrophilic dimer and monomer on autolysis of the extracts, or into a catalytically active amphiphilic monomer by partial reduction. The latter component exists only as trace amounts in the native enzyme. Two additional minor native forms were identified as hydrophilic dimer and monomer. Although a significant proportion of AChE was only solubilized in high salt, following extractions in low salt, this high salt-soluble fraction contained the same molecular forms as the low salt-soluble fractions: thus, we did not detect any molecular form resembling the asymmetric forms of vertebrate cholinesterases.

“…Treatment with proteinase K also led to the loss of the intersubunit disulfide bridge, which resulted in monomeric AChE as seen on SDS-PAGE under nonreducing conditions. This observation parallels that of Landauer et al (1984), but does not concur with DS-AChE from Torpedo marmorata (Stieger and Brodbeck, 1985).…”

Section: Properties Of the Purified Tetrameric Ache Formssupporting

confidence: 50%

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

Gennari

Brodbeck

1985

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.