Detection, resolution, and nomenclature of multiple ubiquitin carboxyl-terminal esterases from bovine calf thymus.
In vivo, ubiquitin exists both free and conjugated through its carboxyl terminus to the alpha- and epsilon-amino groups of a wide variety of cellular proteins. Ubiquitin carboxyl-terminal hydrolytic activity is likely a necessary step in the regeneration of the ubiquitin cofactor from ubiquitin-protein conjugates. In addition, this type of activity is required to generate the active, monomeric ubiquitin from the only known gene products: the polyprotein precursor and various ubiquitin fusion proteins. Thus, this activity is of vital importance to systems that utilize ubiquitin as a cofactor. A generic substrate, ubiquitin ethyl ester, was previously developed [Wilkinson, K. D., Cox, M. J., Mayer, A. N., & Frey, T. (1986) Biochemistry 25, 6644-6649] and utilized here to monitor the fractionation of these activities from calf thymus. By use of a rapid HPLC assay, four distinct, ubiquitin-specific esterases were identified and separated. A previously undescribed activity has been resolved and characterized, in addition to the bovine homologue of ubiquitin carboxyl-terminal hydrolase purified from rabbit reticulocytes. Two other activities resemble deconjugating activities previously detected in crude extracts but not previously purified. These activities appear to form a family of mechanistically related hydrolases. All four activities are inhibited by iodoacetamide, indicating the presence of an essential thiol group, and are inhibited to various extents by manganese. All have specific ubiquitin binding sites as judged by the low observed Km values (0.6-30 microM). The carboxyl-terminal aldehyde of ubiquitin is a potent inhibitor of these enzyme activities, with Ki values approximately 1000-fold lower than the respective Km values.(ABSTRACT TRUNCATED AT 250 WORDS)
Mayer
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AN
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
Wilkinson
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KD
D000818:Animals; D002417:Cattle; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D009626:Terminology as Topic; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013950:Thymus Gland / Q000201:enzymology*; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins / Q000378:metabolism*
D014452:Ubiquitins; D013869:Thiolester Hydrolases; D043222:Ubiquitin Thiolesterase; D007460:Iodoacetamide