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Title: Human brain acetylcholinesterase
Author: Novales-Li, Philipp
ISNI:       0000 0001 3450 0554
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1993
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Procedures for the preparative purification of human acetylcholinesterase (AChE; EC have been examined. Chromatofocussing showed caudate nucleus and cerebrospinal fluid (CSF) AChE eluting between isoelectric points (pi) of 5.2 - 5.57, whilst isoelectric focussing (IEF) revealed heterogeneous AChE isoforms migrating between pI 4.5 - 6. Affinity chromatography proved to be better in yielding highly pure samples. The ligands used were either acridinium or procainamide, which yielded brain AChE recoveries of 10.1% and 42.8%, respectively. Various other methods were used such as gel filtration and ion-exchange chromatography on a Pharmacia SMART System. Purified brain AChE was used as antigen to generate anti-AChE monoclonal antibodies (mAbs) by in vivo immunization. BMS-3E4, -7G10, and -9F4 reacted with erythrocyte (G2) AChE, whilst BMS-6D6 bound to brain (G4) AChE in dot blot, titration, and sedimentation experiments. Conversely, the four mAbs recognized only the G4 form and not the G2 form on immunoblotted IEF and non-denaturing Clarke gels. Deglycosylation studies suggest that the four mAbs recognize a carbohydrate epitope linked to AChE. As a preparative step, tissue culture media containing these mAbs were ammonium sulphate precipitated and purified on a gel filtration column. Under reducing conditions, two bands migrating at 80 and 25 kDa were seen, corresponding to the heavy and light chains of Immunoglobulin M (IgM). Four additional mAbs were raised by the novel method of in vitro immunization, using a synthetically-produced C-terminus peptide as antigen. BMS-5, -6, -7, and -8 are all IgMs which recognize soluble brain and not erythrocyte nor membrane-bound AChE in dot blots, IEF, and sedimentation analyses. Although immunocytochemistry of the above-mentioned mAbs did not demonstrate any positive cholinergic binding, the present mAbs may still offer potential clinical and biochemical applications.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Neurochemistry