title: Improved method for preparation of ubiquitin-ligated lysozyme as substrate of ATP-dependent proteolysis.
abstract: A simple method was developed for preparation of proteins conjugated with ubiquitin. Heat-denatured 125I-labeled lysozyme was highly ubiquitinated by incubation at pH 9.0 with a ubiquitin-protein ligase system consisting of E1, E2 and E3 that had been partially purified from rabbit reticulocytes by affinity chromatography with ubiquitin as a ligand. The resulting conjugates were separated from free lysozyme and other proteins by successive chromatographies on anion and cation ion-exchange resins. The ubiquitinated 125I-lysozymes recovered in the fraction not adsorbed to either resin served as an efficient substrate for ATP-dependent proteolysis in a reticulocyte lysate or with a purified 26 S protease complex. By the present method, 125I-lysozyme-Ub conjugates can be prepared in 3 h with a high yield of 15-20%.
authors.affiliation: Institute for Enzyme Research, University of Tokushima, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Tamura
authors.forename: K
authors.initials: K
authors.lastname: Tanaka
authors.forename: N
authors.initials: N
authors.lastname: Tanahashi
authors.forename: A
authors.initials: A
authors.lastname: Ichihara
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006868:Hydrolysis; D009113:Muramidase / Q000302:isolation & purification*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology; D013379:Substrate Specificity; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D014452:Ubiquitins; D000255:Adenosine Triphosphate; D009113:Muramidase