Isolation of ubiquitin-E2 (ubiquitin-conjugating enzyme) complexes from erythroleukaemia cells using immunoaffinity techniques.
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 (>30 kDa) 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.
Takada
K
K
Department of Biochemistry 1, Jikei University School of Medicine, Nishishinbashi 3-25-8, Minato-ku, Tokyo 105-8461, Japan. takada@jikei.ac.jp
Hirakawa
T
T
Yokosawa
H
H
Okawa
Y
Y
Taguchi
H
H
Ohkawa
K
K
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004952:Esters / Q000302:isolation & purification; D006801:Humans; D020014:K562 Cells; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology*; D008025:Ligases / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D020539:Sequence Analysis, Protein; D013438:Sulfhydryl Compounds / Q000302:isolation & purification; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000302:isolation & purification*
D004952:Esters; D013438:Sulfhydryl Compounds; D014452:Ubiquitins; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases