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 10 different ubiquitin C-terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH-1 was partially purified by conventional chromatographic procedures using (125)I-labeled ubiquitin-alphaNH-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 maximally 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 ubiquitin-alphaNH-protein extensions, including ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids, ubiquitin-alphaNH-dihydrofolate reductase, and poly-His-tagged di-ubiquitin. This enzyme was also capable of generating free ubiquitin from mono-ubiquitin-epsilonNH-protein conjugates and from branched poly-ubiquitin chains that are ligated to proteins through epsilon NH-isopeptide bonds. These results suggest that UCH-1 may play an important role in the generation of free ubiquitin from ubiquitin-ribosomal protein fusions and linear poly-ubiquitin, as well as in recycling of Ub molecules after degradation of poly-ubiquitinated protein conjugates by the 26S proteasome.
Woo
S K
SK
Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Korea.
Baek
S H
SH
Lee
J I
JI
Yoo
Y J
YJ
Cho
C M
CM
Kang
M S
MS
Chung
C H
CH
D000595:Amino Acid Sequence; D000818:Animals; D019759:Carbon-Nitrogen Lyases; D002645:Chickens; D002845:Chromatography; D004586:Electrophoresis; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007460:Iodoacetamide / Q000494:pharmacology; D008190:Lyases / Q000378:metabolism; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins / Q000378:metabolism
D007457:Iodine Radioisotopes; D010455:Peptides; D014452:Ubiquitins; D013869:Thiolester Hydrolases; D043222:Ubiquitin Thiolesterase; D008190:Lyases; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase; D007460:Iodoacetamide