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dc.contributor.authorVaughan, Roxanne Louiseen
dc.date.accessioned2014-08-13T14:38:51Zen
dc.date.available2014-08-13T14:38:51Zen
dc.date.issued1985en
dc.identifier.urihttp://hdl.handle.net/10919/49950en
dc.description.abstractA developmentally regulated cyclic AMP-dependent protein kinase has been recently reported in Dictyostelium discoideum. This report describes some of the physical and kinetic properties of the cAMP-dependent holoenzyme and its subunits. Gel filtration data suggests a holoenzyme Mr of 170,000-190,000, and catalytic and regulatory subunit Mrs of 40,000 and 49,000, respectively. These molecular weight determinations are compatible with an R₂C₂ subunit arrangement of the holoenzyme. Kinase activity required the presence of Mg²⁺ but cAMP binding to the enzyme was not dependent on divalent metal ions. The pH optimum for kinase activity was 7.5; the cAMP binding activity was not affected over a pH range of 5.0-10.0. The holoenzyme and isolated regulatory subunit had identical cAMP Kds of 28 nM. Cyclic AMP was able to dissociate the subunits when analyzed by density gradient centrifugation. Histone VII-S activated the subunits in the absence of cAMP but did not produce their dissociation. In contrast to the gel filtration data, sedimentation values indicated a dimeric holoenzyme structure. Reassociation of the subunits in the absence of cAMP occurred rapidly and was not dependent upon a preincubation with MgATP. High NaCl and low pH depressed both the total kinase activity and the ability of the subunits to reassociate as determined by activity ratio. MgATP did not decrease the ability of the holoenzyme to bind cAMP, neither did the holoenzyme possess a high affinity MgATP binding site. By the use of microdissection techniques holoenzyme levels were determined in individuals at each stage of development and in each cell type during development. Kinase activity was low and non-cAMP dependent in early aggregates but increased and became cAMP-dependent in later aggregates. Maximum activity and cAMP-dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single-stage occurred during culmination when the activity in the stalks was approximately one-fourth that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor.en
dc.format.extentix, 125 leavesen
dc.format.mimetypeapplication/pdfen
dc.publisherVirginia Polytechnic Institute and State Universityen
dc.relation.isformatofOCLC# 12888773en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subject.lccLD5655.V856 1985.V387en
dc.subject.lcshDictyostelium discoideum -- Developmenten
dc.subject.lcshDictyostelium discoideum -- Cytologyen
dc.subject.lcshCell differentiationen
dc.subject.lcshCyclic adenylic aciden
dc.subject.lcshEnzymatic analysisen
dc.titleCharacterization and localization of a cyclic AMP dependant protein kinase from Dictyostelium discoideumen
dc.typeDissertationen
dc.contributor.departmentZoologyen
dc.description.degreePh. D.en
thesis.degree.namePh. D.en
thesis.degree.leveldoctoralen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.disciplineZoologyen
dc.type.dcmitypeTexten
dc.description.adminincomplete_metadataen


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