Purification of E1 and E1-like enzymes.
Ubiquitin-activating enzyme is the archetype for a family of enzymes catalyzing the ATP-coupled activation of ubiquitin and other class 1 ubiquitin-like proteins required for their subsequent conjugation to cellular targets. The general physical and mechanistic features of the E1 family appear well conserved. Formation of an obligatory E1-ubiquitin thiol ester intermediate forms the basis of a one-step covalent purification of the enzyme on ubiquitin-linked affinity columns that has been adapted for the isolation of E1 paralogs. We describe the facile purification of active E1 from outdated human red blood cells in yields (2-4 nmol/U of blood) that make this an attractive alternative to expression of the proteolytically labile recombinant protein. In addition, two stoichiometric activity assays are described that rely on formation of the E1 125I-ubiquitin thiol ester and ubiquitin [2,8-3H]adenylate intermediates.
Haas
Arthur L
AL
Department of Biochemistry and Molecular Biology, Louisiana State University Health Science Center, New Orleans, USA.
D002846:Chromatography, Affinity / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry / Q000302:isolation & purification*; D014452:Ubiquitins / Q000737:chemistry*
D014452:Ubiquitins; D044764:Ubiquitin-Activating Enzymes