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dc.contributor.authorChevesich, Jorge Alejandro
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 54-01, Section: B, page: 2150.;Advisors: Umadas Maitra.
dc.description.abstractEukaryotic translation initiation factor 5 (eIF-5) promotes the hydrolysis of GTP bound to the 40S initiation complex (40S{dollar}\cdot{dollar}mRNA{dollar}\cdot{dollar}Met-tRNA{dollar}\sb{lcub}\rm f{rcub}{lcub}\cdot{rcub}{dollar}eIF-2{dollar}{lcub}\cdot{rcub} \rm GTP){dollar}. The eIF-2{dollar}\cdot{dollar}GDP and P{dollar}\sb{lcub}\rm i{rcub}{dollar} formed in the reaction are released from the 40S ribosomal subunit. The resulting 40S complex (40S{dollar}\cdot{dollar}mRNA{dollar}\cdot{dollar}Met-tRNA{dollar}\sb{lcub}\rm f{rcub}){dollar} then joins a 60S ribosomal subunit in the absence of eIF-5 to form an elongation-competent 80S ribosomal initiation complex. Using an assay that directly measure the formation of {dollar}\sp{lcub}32{rcub}\rm P\sb{lcub}i{rcub}{dollar} from ({dollar}\gamma{dollar}-{dollar}\sp{lcub}32{rcub}{dollar}P) GTP bound to the 40S initiation complex, a new and rapid procedure for the isolation and purification of eIF-5 from rabbit reticulocyte lysates if presented. The procedure described leads to the isolation of eIF-5 that is not only physically homogeneous, as judged by electrophoretic analysis in polyacrylamide gels, but also, more importantly, was obtained in 15-20 times greater yield than that previously reported from this and other laboratories. Analysis of the size of the purified protein by glycerol gradient centrifugation as well as by SDS-polyacrylamide gel electrophoresis demonstrates that purified eIF-5 is a monomeric protein of about 58 kDa. In contrast, less pure preparations of reticulocyte eIF-5 behave in glycerol gradient centrifugation in buffers containing 100 mM KCl as a protein of about 160-kDa. Presumably, this is due to association of the factor with other proteins. Additional characterization of eIF-5 presented in this thesis indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. However, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the formation of an 80S initiation complex from a preformed 40S initiation complex.;Immunochemical methods have been employed to characterize the in vivo form of eIF-5. Antibodies against purified native eIF-5 were isolated from egg yolks of laying hens immunized with reticulocyte eIF-5 and characterized by immunoblotting and immunoprecipitation techniques using denatured and native eIF-5 as antigens. When the GH{dollar}\sb3{dollar} cells are labeled with {dollar}\sp{lcub}32{rcub}\rm P\sb{lcub}i{rcub}{dollar} and eIF-5 is isolated from these cells by immunoprecipitation followed by denaturing gel electrophoresis, the factor is found to be phosphorylated at serine residues. Phosphopeptide mapping of in vivo {dollar}\sp{lcub}32{rcub}{dollar}P-labeled eIF-5 shows two major sites of phosphorylation that are distinct from the rabbit reticulocyte eIF-5 sites phosphorylated in vitro by casein kinase II. The biological significance of specific phosphorylation of eIF-5 is discussed.
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.subjectCellular biology.
dc.titleCharacterization of mammalian translation initiation factor 5 in vitro and in vivo

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