Proteasome activator subunit PA28α and related Ki antigen (PA28γ) are absent from the nuclear fraction purified by sucrose gradient centrifugation

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Abstract

The aim of the present work was to attempt to partially purify PA28 (REG) α and γ (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-β-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. PA28α and γ were detected mainly in the cytosol and to a lesser extent in the crude nuclear pellet, however the purified nuclei were devoid of PA28α and γ. 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.

Original languageEnglish (US)
Pages (from-to)273-276
Number of pages4
JournalInternational Journal of Biochemistry and Cell Biology
Volume31
Issue number2
DOIs
StatePublished - Feb 1999
Externally publishedYes

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Centrifugation
Proteasome Endopeptidase Complex
Chromatin
Sucrose
Nuclear Envelope
Tubulin
Cytosol
Histones
Purification
Fluorescent Antibody Technique
Cytoplasm
Western Blotting
Monoclonal Antibodies
Membranes
Ki antigen

Keywords

  • NT2 cells
  • Nucleus
  • PA28
  • Proteasome
  • Proteasome activator

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

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title = "Proteasome activator subunit PA28α and related Ki antigen (PA28γ) 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) α and γ (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-β-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. PA28α and γ were detected mainly in the cytosol and to a lesser extent in the crude nuclear pellet, however the purified nuclei were devoid of PA28α and γ. 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.",
keywords = "NT2 cells, Nucleus, PA28, Proteasome, Proteasome activator",
author = "Cezary Wojcik",
year = "1999",
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TY - JOUR

T1 - Proteasome activator subunit PA28α and related Ki antigen (PA28γ) are absent from the nuclear fraction purified by sucrose gradient centrifugation

AU - Wojcik, Cezary

PY - 1999/2

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N2 - The aim of the present work was to attempt to partially purify PA28 (REG) α and γ (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-β-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. PA28α and γ were detected mainly in the cytosol and to a lesser extent in the crude nuclear pellet, however the purified nuclei were devoid of PA28α and γ. 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.

AB - The aim of the present work was to attempt to partially purify PA28 (REG) α and γ (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-β-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. PA28α and γ were detected mainly in the cytosol and to a lesser extent in the crude nuclear pellet, however the purified nuclei were devoid of PA28α and γ. 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.

KW - NT2 cells

KW - Nucleus

KW - PA28

KW - Proteasome

KW - Proteasome activator

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