Proteasome activator subunit PA28 alpha and related Ki antigen (PA28 gamma) are absent from the nuclear fraction purified by sucrose gradient centrifugation.
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.
Wójcik
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C
Department of Histology and Embryology, Warsaw Medical Academy, Poland. cwojcik@ib.amwaw.edu.pl
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
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