Show simple item record

dc.contributor.authorChou, Jordan
dc.contributor.otherQueen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.))en
dc.date2010-10-25 16:03:52.364en
dc.date.accessioned2010-10-25T21:01:48Z
dc.date.available2010-10-25T21:01:48Z
dc.date.issued2010-10-25T21:01:48Z
dc.identifier.urihttp://hdl.handle.net/1974/6172
dc.descriptionThesis (Master, Biochemistry) -- Queen's University, 2010-10-25 16:03:52.364en
dc.description.abstractCalpains are calcium-dependent, intracellular, multi-domain cysteine proteases involved in many physiological functions regulated by calcium signaling, including cell motility. How calpains are activated in the cell is still unknown because the resting intracellular concentration of Ca2+ is orders of magnitude lower than that needed for half-maximal activation of the enzyme in vitro. Several stratagems by which calpains might overcome this Ca2+ concentration differential have been proposed. It is possible that post-translational modifications like phosphorylation, or accessory proteins that bind to calpain, might facilitate the enzyme’s activation at lower than optimal Ca2+ concentrations. Autoproteolysis (autolysis) and subunit dissociation are two other proposed activation mechanisms that could release constraints on the calpain core by breaking the link between the anchor helix and the small subunit to allow the active site to form. By measuring the rate of autolysis at different sites in calpain, it was demonstrated that while the anchor helix is one of the first targets to be cut, several other potentially inactivating autolysis sites, particularly in Domain III, can also be cleaved within the first minute. Thus autolytic activation would go hand in hand with inactivation. By fractionating and identifying calpain 2 autolysis fragments, I show that the small subunit does not dissociate away from the large subunit, but is proteolyzed to a 40-45 k heterodimer of the penta-EF-hand Domains IV and VI. It is likely that this autolysis-generated heterodimer has previously been misidentified as the small subunit domain VI homodimer that would be produced by subunit dissociation. A calpastatin affinity column was constructed and used to capture recombinant calpain 2 from bacterial cell lysate. This affinity column provides a tool to screen for and capture calpain complexed to potential binding partners in the presence of Ca2+. Here I propose a model for calpain 2 activation in vitro that does not involve autolysis, subunit dissociation, or calpain activators.en
dc.languageenen
dc.language.isoenen
dc.relation.ispartofseriesCanadian thesesen
dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.subjectCalpainen
dc.subjectActivation Modelen
dc.subjectAutolysisen
dc.subjectSubunit Dissociationen
dc.subjectMass Spectrometryen
dc.subjectChromatographyen
dc.subjectCalciumen
dc.titleCALPAIN 2 ACTIVATION, AUTOLYSIS, AND SUBUNIT DISSOCIATIONen
dc.typeThesisen
dc.description.degreeMasteren
dc.contributor.supervisorDavies, Peter L.en
dc.contributor.departmentBiochemistryen


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record