title: Proteasome activator subunit PA28 alpha and related Ki antigen (PA28 gamma) are absent from the nuclear fraction purified by sucrose gradient centrifugation.
abstract: The aim of the present work was to attempt to partially purify PA28 (REG) alpha and gamma (Ki antigen) in the nuclear fraction from NT2/D1 cells. Nuclei were isolated by the hypertonic sucrose gradient centrifugation method and fractionated into membrane/nucleoplasmic and chromatin/nucleolar fractions. Western blotting with anti-histone and anti-beta-tubulin monoclonal antibodies confirmed the accuracy of the procedure. Proteasomes were present mainly in the cytoplasm but also in the nuclei. Disruption of the nuclear envelope released the proteasomes implying a loose or no binding with the chromatin. PA28 alpha and gamma were detected mainly in the cytosol and to a lesser extent in the crude nuclear pellet, however the purified nuclei were devoid of PA28 alpha and gamma. This indicates, that only a small fraction of the PA28 activator is present in the nuclei as detected by immunofluorescence or/and it is easily removed during nuclear purification.
authors.affiliation: Department of Histology and Embryology, Warsaw Medical Academy, Poland. cwojcik@ib.amwaw.edu.pl
authors.forename: C
authors.initials: C
authors.lastname: Wójcik
mesh_terms: D000954:Antigens, Surface / Q000302:isolation & purification / Q000378:metabolism; D001324:Autoantigens; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000379:methods*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism; D009124:Muscle Proteins; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D013347:Subcellular Fractions; D013395:Sucrose / Q000737:chemistry; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000954:Antigens, Surface; D001324:Autoantigens; C099558:Ki antigen; D009097:Multienzyme Complexes; D009124:Muscle Proteins; D009687:Nuclear Proteins; C085047:PSME1 protein, human; D011506:Proteins; D014404:Tubulin; D013395:Sucrose; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex