A Novel UbiquitinC‐terminal Hydrolase (UCH‐9) from Chick Skeletal Muscle: Its Purification and Characterization

Abstract

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C‐terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH‐9, was purified by conventional Chromatographie procedures using 125I‐Iabeled ubiquitin‐αNH‐MHISPPEPESEEEEE‐HYC (Ub‐PESTc) as a substrate. The purified enzyme behaved as a 27‐kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It was maximally active at pHs between 7 and 8.5, but showed little or no activity at pH below 6 and above 10. Like other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide, and by Ub‐aldehyde. In addition to Ub‐PESTc, UCH‐9 hydrolyzed Ub‐αNH‐protein extensions, including Ub‐αNH‐carboxyl extension protein of 80 amino acids and Ub‐αNH‐dihydrofolate reductase. However, this enzyme was not capable of generating free Ub from mono‐Ub‐ϵNH‐protein conjugates and from branched poly‐Ub chains that are ligated to proteins through ϵNH‐isopeptide bonds. This enzyme neither could hydrolyze poly‐His‐tagged di‐Ub. These results suggest that UCH‐9 may play an important role in production of free Ub and ribosomal proteins from their conjugates.