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dc.contributor.authorGough, Nancy Redmond
dc.date.accessioned2013-04-03T17:06:22Z
dc.date.available2013-04-03T17:06:22Z
dc.date.issued1993
dc.identifier.urihttp://hdl.handle.net/10713/2490
dc.descriptionUniversity of Maryland, Baltimore. Pharmacology. Ph.D. 1993en_US
dc.description.abstractAcetylcholinesterase (AChE) exists in multiple molecular forms: soluble and amphiphilic oligomers and membrane-bound forms. Mechanisms for the generation of this molecular diversity of AChE in chicken were investigated. No evidence for the diversity in chicken AChE arising from alternative splicing of coding exons was found. Generation of the molecular forms of AChE from a single transcript was studied by transfecting plasmids encoding the open reading frame of the T subunit of chicken AChE into a human kidney fibroblast cell line (293) and a mouse muscle cell line {dollar}\rm(C\sb2C\sb{lcub}12{rcub}).{dollar} The subcellular localization and biosynthesis of the molecular forms of the transfected protein were determined. In both cell lines, a fraction of the expressed protein was targeted to the membrane despite the absence of a traditional membrane spanning domain. The role of ionic and hydrophobic interactions in membrane binding was studied using liposomes of defined lipid composition. The importance of the C-terminus of the enzyme in membrane association and homo-oligomerization was analyzed in the transfected 293 cells using site-directed mutagenesis of the C-terminus. The C-terminal cysteine is the only cysteine not involved in intramolecular disulfide bonding. Chicken AChE can bind to the membrane without the C-terminal cysteine or C-terminal 39 amino acids. The protein can also form homo-oligomers without the C-terminal cysteine or the last 39 amino acids. The ability to form oligomeric AChE was not a prerequisite for membrane association, since monomeric AChE was detected in membrane extracts. Mutant enzymes lacking the only free cysteine, cys{dollar}\sb{lcub}764{rcub},{dollar} formed oligomers and targeted to the membrane indicating that the oligomeric AChE does not require disulfide bonds for stabilization nor is membrane binding mediated through covalent attachment of a second protein by disulfide bonds. AChE from cells transfected with the wild-type or mutant constructs were labeled with tritiated palmitate suggesting that fatty acylation may be involved in membrane association. A region other than the C-terminus contains the signal for targeting to the membrane and the site for palmitoylation. Oligomeric chicken AChE is stabilized through hydrophobic interactions of the C-terminus and does not require intersubunit disulfide bonds.en_US
dc.language.isoen_USen_US
dc.subjectBiology, Molecularen_US
dc.subjectHealth Sciences, Pharmacologyen_US
dc.subject.meshAcetylcholinesterase--analysisen_US
dc.subject.meshAcetylcholinesterase--biosynthesisen_US
dc.subject.meshChickensen_US
dc.titleThe biosynthesis of the molecular forms of chicken acetylcholinesteraseen_US
dc.typedissertationen_US
dc.contributor.advisorRandall, William R., Ph.D.
dc.identifier.ispublishedYes
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