PURIFICATION AND CHARACTERIZATION OF A NEW UBIQUITIN C-TERMINAL HYDROLASE (UCH-1) WITH ISOPEPTIDASE ACTIVITY FROM CHICK SKELETAL-MUSCLE

we have previously shown that chick muscle extracts contain at least 1 0 different ubiquitin C-terminal hydrolases (UCHs), In the present stu dies, one of the enzymes, called UCH-1 was partially purified by conve ntional chromatographic procedures using I-125-labeled ubiquitin- alph a NH-MHISPPEPESEEEEEHYC as a substrate, The purified enzyme behaved as a 35-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consisted of a single polypeptide chain, It was max imally active at pHs between 8 and 9 but showed little or no activity at pH below 6 and above 11. Like other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide and by ubiquitin-aldehyde, In addition to Ub -PESTc, UCH-1 hydrolyzed ubiq uitin- alpha NH-protein extensions, including ubiquitin-alpha NH-carbo xyl extension protein of 80 amino acids ubiquitin- alpha NH-dihydrofol ate reductase, and poly-sis-tagged di-ubiquitin. This enzyme was also capable of generating free ubiquitin from mono-ubiquitin- epsilon NH-p rotein conjugates and from branched poly-ubiquitin chains that are lig ated to proteins through epsilon NH-isopeptide bonds, These results su ggest that UCH-1 may play an important role in the generation of free ubiquitin from ubiquitin-ribosomal protein fusions and linear polyubiq uitin, as well as in recycling of Ub molecules after degradation of po ly-ubiquitinated protein conjugates by the 265 proteasome.