"Covalent affinity" purification of ubiquitin-activating enzyme.
We have previously described an enzyme that activates ubiquitin, the heat-stable polypeptide of the ATP-dependent proteolytic system from reticulocytes (Ciechanover, A., Heller, H., Katz-Etzion, R., and Hershko, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 761-765). It carries out ubiquitin-dependent PPi-ATP and AMP-ATP exchange reactions and binds to the activated polypeptide by a thiolester linkage. We describe here a procedure for the purification of this enzyme by its binding to ubiquitin-Sepharose. Binding of the enzyme to the affinity column requires ATP and Mg2+, and bound enzyme cannot be displaced by high salt but can be eluted by raising the pH, by increased concentrations of a thiol compound, or by the joint supplementation of AMP and pyrophosphate. Another form of the enzyme that cannot carry out AMP-ATP exchange (but catalyzes ubiquitin-dependent PPi-ATP exchange) does not bind to the affinity column. These data indicate that a covalent, possibly thiolester intermediate, is formed between the activating enzyme and Sepharose-bound ubiquitin. It is suggested designating this procedure of enzyme isolation "covalent affinity" chromatography. The purified enzyme has an apparent Mr = 210,000 and appears to be composed of two subunits of Mr = 105, 000. ATP-dependent binding of ubiquitin to the purified enzyme and to its subunit is demonstrated.
D000818:Animals; D002846:Chromatography, Affinity; D002868:Chromosomal Proteins, Non-Histone / Q000097:blood / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification* / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D009698:Nucleoproteins / Q000097:blood; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D013379:Substrate Specificity; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins
D002868:Chromosomal Proteins, Non-Histone; D046911:Macromolecular Substances; D009698:Nucleoproteins; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes