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Research article

Isolation of ubiquitin–E2 (ubiquitin-conjugating enzyme) complexes from erythroleukaemia cells using immunoaffinity techniques

Koji TAKADA, Tae HIRAKAWA, Hideyoshi YOKOSAWA, Yutaka OKAWA, Hideki TAGUCHI, Kiyoshi OHKAWA
Biochemical Journal May 15, 2001, 356 (1) 199-206; DOI: 10.1042/bj3560199
Koji TAKADA
Department of Biochemistry 1, Jikei University School of Medicine, Nishishinbashi 3-25-8, Minato-ku, Tokyo 105-8461, Japan
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Tae HIRAKAWA
Department of Biochemistry 1, Jikei University School of Medicine, Nishishinbashi 3-25-8, Minato-ku, Tokyo 105-8461, Japan
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Hideyoshi YOKOSAWA
Department of Biochemistry, Graduate School of Pharmaceutical Science, Hokkaido University, Sapporo 060-0812, Japan
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Yutaka OKAWA
Department of Biochemistry 1, Jikei University School of Medicine, Nishishinbashi 3-25-8, Minato-ku, Tokyo 105-8461, JapanDepartment of Internal Medicine, Jikei University School of Medicine, Nishishinbashi 3-25-8, Minato-ku, Tokyo 105-8461, Japan
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Hideki TAGUCHI
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama, 226-8503, Japan
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Kiyoshi OHKAWA
Department of Biochemistry 1, Jikei University School of Medicine, Nishishinbashi 3-25-8, Minato-ku, Tokyo 105-8461, Japan
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Abstract

A variety of ubiquitin-associated (or conjugated) proteins, including substrates and enzymes for the ubiquitin system, are present in eukaryotic cells. In the present study we developed a simple method for their isolation, consisting of immunoaffinity chromatography using the monoclonal antibody FK2, which recognizes the conjugated ubiquitin molecule. Using this method followed by gel filtration, we isolated multi-ubiquitinated proteins with high molecular masses (> 30kDa) and also ubiquitinthioester-linked and mono-ubiquitinated forms of ubiquitin-conjugating (E2) enzymes, UbcH7 and UBE2N, together with mono-, di- and tri-ubiquitin molecules, from the cytoplasmic extract of heat-shock-treated K562 erythroleukaemia cells. We also demonstrated that the FK2 antibody was capable of precipitating a ubiquitin–UbcH7 thioester, but not free UbcH7, which enabled the measurement of the respective cellular levels separately. The immunoprecipitable ubiquitin–UbcH7 thioester was found only when the cells were treated with heat-shock. These results suggest the usefulness of the immunoaffinity techniques for identifying and analysing the cellular enzyme/protein–ubiquitin complexes.

  • affinity chromatography
  • heat-shock
  • immunoprecipitation
  • thioester

Footnotes

  • Abbreviations used: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; MUCRP1, multi-ubiquitin chain reference preparation 1; DTT, dithiothreitol; MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS.

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May 2001

Volume: 356 Issue: 1

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Isolation of ubiquitin–E2 (ubiquitin-conjugating enzyme) complexes from erythroleukaemia cells using immunoaffinity techniques
Koji TAKADA, Tae HIRAKAWA, Hideyoshi YOKOSAWA, Yutaka OKAWA, Hideki TAGUCHI, Kiyoshi OHKAWA
Biochemical Journal May 2001, 356 (1) 199-206; DOI: 10.1042/bj3560199
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Isolation of ubiquitin–E2 (ubiquitin-conjugating enzyme) complexes from erythroleukaemia cells using immunoaffinity techniques
Koji TAKADA, Tae HIRAKAWA, Hideyoshi YOKOSAWA, Yutaka OKAWA, Hideki TAGUCHI, Kiyoshi OHKAWA
Biochemical Journal May 2001, 356 (1) 199-206; DOI: 10.1042/bj3560199

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Keywords

affinity chromatography
heat-shock
immunoprecipitation
thioester

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