• Login as Editor
    View Item 
    •   Yeshiva Academic Institutional Repository
    • Albert Einstein College of Medicine (AECOM)
    • Albert Einstein College of Medicine: Doctoral Dissertations
    • View Item
    •   Yeshiva Academic Institutional Repository
    • Albert Einstein College of Medicine (AECOM)
    • Albert Einstein College of Medicine: Doctoral Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Characterization of mammalian translation initiation factor 5 in vitro and in vivo

    Thumbnail
    Date
    1993
    Author
    Chevesich, Jorge Alejandro
    Metadata
    Show full item record
    Abstract
    Eukaryotic 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.
    Permanent Link(s)
    https://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9314334
    https://hdl.handle.net/20.500.12202/3476
    Collections
    • Albert Einstein College of Medicine: Doctoral Dissertations [1674]

    Yeshiva University Libraries copyright © 2021  DuraSpace
    YAIR Self-Deposit | YAIR User's Guide | Take Down Policy | Contact Us
    Yeshiva University
     

     

    Browse

    AllCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects

    My Account

    Login as Editor

    Statistics

    View Usage Statistics

    Yeshiva University Libraries copyright © 2021  DuraSpace
    YAIR Self-Deposit | YAIR User's Guide | Take Down Policy | Contact Us
    Yeshiva University