New de-ubiquitinating enzyme, ubiquitin C-terminal hydrolase 8, in chick skeletal muscle.
We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). Here we report the purification and characterization of one of the UCHs, called UCH-8, with 125I-labelled ubiquitin-alpha-NH-MHISPPEPESEEEEEHYC as a substrate. The purified UCH-8 behaved as a 240 kDa protein on a Superdex-200 column under non-denaturing conditions but as a 130 kDa polypeptide on analysis by PAGE under denaturing conditions, suggesting that the enzyme consists of two identical subunits. Thus this enzyme seems to be distinct in its dimeric nature from other purified UCHs that consist of a single polypeptide, except that UCH-6 is also a homodimer of 27 kDa subunits. UCH-8 was maximally active between pH 7.5 and 8, but showed little or no activity below pH 7 and above pH 9. Like other UCHs it was sensitive to inhibition by thiol-blocking agents such as N-ethylmaleimide, and by ubiquitin aldehyde. The purified UCH-8 hydrolysed not only ubiquitin-alpha-NH-protein extensions, including ubiquitin-alpha-NH-carboxy extension protein of 80 amino acid residues and ubiquitin-alpha-NH-dihydrofolate reductase, but also branched poly-ubiquitin that are ligated to proteins through epsilon-NH-isopeptide bonds. However, it showed little or no activity against poly-His-tagged di-ubiquitin, suggesting that UCH-8 is not involved in the generation of free ubiquitin from the linear poly-ubiquitin precursors. These results suggest that UCH-8 might have an important role in the production of free ubiquitin and ribosomal proteins from their conjugates as well as in the recycling of ubiquitin molecules after the degradation of poly-ubiquitinated protein conjugates by the 26 S proteasome.
Baek
S H
SH
Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea.
Woo
S K
SK
Lee
J I
JI
Yoo
Y J
YJ
Cho
C M
CM
Kang
M S
MS
Tanaka
K
K
Chung
C H
CH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002412:Cations / Q000494:pharmacology; D002645:Chickens; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010369:Pectoralis Muscles / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011107:Polylysine / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000906:Antibodies; D002412:Cations; D007527:Isoenzymes; D010455:Peptides; D011480:Protease Inhibitors; D011107:Polylysine; D013869:Thiolester Hydrolases; C107388:ubiquitin C-terminal hydrolase 8