Isolation and characterization of cytosolic and membrane-bound deubiquitinylating enzymes from bovine brain.
The deubiquitinylating enzymes (DUBs), that release free ubiquitin (Ub) from its precursors or ubiquitinylated proteins, are known to comprise of a large protein family in eukaryotes, but those in mammalian tissues remain largely unknown. Here we report the existence of unexpectedly large species of DUBs in both soluble and membrane-bound fractions of bovine brain, based on their ability to cleave (125)I-labeled Ub-fused alphaNH-MHISPPEPESEEEEEHYC (designated as Ub-PESTc). Two cytosolic enzymes, tentatively called sDUB-1 and sDUB-2, with molecular masses of about 30 kDa were purified to near homogeneity by Ub-Sepharose affinity chromatography. sDUB-1 and sDUB-2 corresponded to UCH-L3 and UCH-L1/PGP 9.5, respectively. Intriguingly, the particulate fraction of the brain homogenate was found to also contain strong activities against (125)I-Ub-PESTc, which can be solubilized by treatment with 5% n-heptyl-beta-D-thioglucoside and 1% Nonidet P-40, but not by washing with 1 M NaCl. From the solubilized material, two new 30-kDa, membranous DUBs (called mDUB-1 and mDUB-2) were purified to apparent homogeneity by Ub-Sepharose chromatography. Two other Ub-aldehyde sensitive DUBs, designated as mDUB-3 and mDUB-4, were also partially purified by conventional chromatographic operations. These mDUBs differed from each other in substrate specificity and exhibited different characteristics from the sDUBs, revealing that they are a new type of membrane-bound DUB. These results indicate the presence of divergent DUBs in mammalian brain, which may contribute to regulation of numerous pivotal cellular functions mediated by the covalent modification of Ub.
Kawakami
T
T
Department of Gastroenterology, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8655, Japan.
Suzuki
T
T
Baek
S H
SH
Chung
C H
CH
Kawasaki
H
H
Hirano
H
H
Ichiyama
A
A
Omata
M
M
Tanaka
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013869:Thiolester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases
D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D013869:Thiolester Hydrolases; D010450:Endopeptidases; D064570:Ubiquitin-Specific Proteases