Reconstituting ubiquitination reactions with affinity-purified components and 32P-ubiquitin.
The discovery of protein ubiquitination in a broad range of organisms and experimental settings has raised the need for a straightforward way to characterize the mechanism of substrate targeting, using purified components. The mechanism of ubiquitin conjugation to proteins has been extensively studied and is mediated by a family of evolutionarily conserved proteins. We have used previously described expression systems to purify the relevant targeting components of the ubiquitin system. These methods yielded substantial amounts of highly purified and catalytically active enzymes that permitted their use in reconstituting protein ubiquitination. We monitored ubiquitination reactions with 32P-ubiquitin rather than 125I-ubiquitin. This advance makes the procedure accessible to a broader range of experimentalists, since it eliminates the additional concerns involved in handling 125I-isotope. Furthermore, the strategies described here can be used to investigate the effects of specific mutations introduced into ubiquitin or the targeting components (E1, Ubc/E2, and E3) of this pathway.
Tongaonkar
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P
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, New Jersey, 08854, USA. maduraki@umdnj.edu
Madura
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K
D000595:Amino Acid Sequence; D000818:Animals; D005075:Biological Evolution; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D017124:Conserved Sequence; D017931:DNA Primers; D005982:Glutathione Transferase / Q000378:metabolism; D007457:Iodine Radioisotopes; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000379:methods; D011865:Radioisotope Dilution Technique; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D007457:Iodine Radioisotopes; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D008025:Ligases