Affinitätschromatographische Reinigung der Acetylcholinesterase aus Nucleus Caudatus vom Rind im Großmaßstab

Ruess, Klaus-Peter; Weinert, M.; Liefländer, Manfred

doi:10.1515/bchm2.1976.357.1.783

Cited by 18 publications

(7 citation statements)

References 11 publications

(5 reference statements)

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“…1C). The maximal specific activity of cell-associated AChE of approximately 0.1 IU/mg protein is about three times lower than that found in huiman (Ssrensen et al, 1982) and bovine caudate nucleus (Ruess et al, 1976), but 5-10 times higher than that in all human neuroblastoma cell lines tested so far (result not shown). The increase in AChE per day and per milligram of cell protein was similar for cell-associated and secreted enzyme and it was nearly independent of the state of cell growth (Fig.…”

Section: Production Of Cellular and Secreted Achementioning

confidence: 63%

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

Stieger

Bütikofer

Wiesmann

et al. 1989

Journal of Neurochemistry

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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.

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Section: Production Of Cellular and Secreted Achementioning

confidence: 63%

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

Stieger

Bütikofer

Wiesmann

et al. 1989

Journal of Neurochemistry

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“…As previously mentioned, hydrophobic AChE has also been purified to homogeneity from the brains of a number of vertebrate species, including bovine [121], rat [122, 1231, human [124] and chicken [125]. In all these cases the purified brain enzyme occurs as a G4 tetramer.…”

Section: Globular Forms Purification Characterization and Hydrophobimentioning

confidence: 99%

Modes of attachment of acetylcholinesterase to the surface membrane

Silman

Futerman

1987

European Journal of Biochemistry

173

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Acetylcholinesterase (AChE) occurs in multiple molecular forms differing in their quaternary structure and mode of anchoring to the surface membrane. Attachment is achieved by post‐translational modification of the catalytic subunits. Two such mechanisms are described. One involves attachment to catalytic subunit tetramers, via disulfide bridges, of a collagen‐like fibrous tail. This, in turn, interacts, primarily via ionic forces, with a heparin‐like proteoglycan in the extracellular matrix. A second such modification involve the covalent attachment of a single phosphatidylinositol molecule at the carboxyl‐terminus of each catalytic subunit polypeptide; the diacylglycerol moiety of the phospholipid serves to anchor the modified enzyme hydrophobically to the lipid bilayer of the plasma membrane. The detailed molecular structure of these two classes of acetylcholinesterase are discussed, as well as their biosynthesis and mode of anchoring.

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“…Solubilization of AChE from bovine NC membranes, purification by affinity chromatography, and removal of detergent from the purified 10.5 S AChE/Triton X-100 complex were done according to the methods described originally by Ruess et al (1976), with the modification that the cytoplasmic enzyme was removed by centrifugation (53,000 g for 3 h) prior to solubilization of the membrane-bound enzyme. AChE preparations with specific activities varying between 4,000 and 4,250 U per milligram protein were obtained.…”

Section: Enzyme Purificationmentioning

confidence: 99%

“…Brain acetylcholinesterase (AChE, EC 3.1.1.7) from five different mammalian sources has been purified to homogeneity, with specific activities ranging from 3,000 to 4,500 Ulmg (Wenthold et al, 1974;Ruess et al, 1976;Greenberg et al, 1977;Rakonczay et al, 1981;Sgirensen et al, 1982). It is well established that the molecular forms of AChE from brain tissues are globular forms (G-forms) and occur in two main classes-nonhydrophobic and hydrophobic-differing in solubility properties, aggregation behavior, and the ability to interact with arnphiphilic molecules Landauer et al, 1982;Sgirensen et al, 1982).…”

mentioning

confidence: 99%

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

Landauer¹,

Ruess²,

Liefländer³

1984

Journal of Neurochemistry

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The number of catalytic subunits of purified bovine nucleus caudatus acetylcholinesterase (E.C. 3.1.1.7) has been determined by active site labelling with [3H]diisopropyl fluorophosphate ([3H]DFP). The 10.5 S, 16 S, and 20 S forms were estimated to contain two, four, and six active sites, respectively, per molecule. A 4.8 S form, which showed a weak amphiphile-dependent activity behavior, was obtained by selective proteolytic digestion with pronase. The inability of the purified 4.8 S form to aggregate after detergent removal, and the molecular mass in the range of 130-165 kD under nondenaturating conditions, indicate that this form is a dimeric form, lacking those hydrophobic regions responsible for aggregation.

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Affinitätschromatographische Reinigung der Acetylcholinesterase aus Nucleus Caudatus vom Rind im Großmaßstab

Cited by 18 publications

References 11 publications

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

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

Modes of attachment of acetylcholinesterase to the surface membrane

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

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Affinitätschromatographische Reinigung der Acetylcholinesterase aus Nucleus Caudatus vom Rind im Großmaßstab

Cited by 18 publications

References 11 publications

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

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

Modes of attachment of acetylcholinesterase to the surface membrane

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

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

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