Isolation and characterization of cytosolic and membrane-bound deubiquitinylating enzymes from bovine brain

The deubiquitinylating enzymes (DUBs), that release free ubiquitin (Ub) fro m its precursors or ubiquitinylated proteins, are known to comprise of a la rge protein family in eukaryotes, but those in mammalian tissues remain lar gely unknown. Here we report the existence of unexpectedly large species of DUBs in both soluble and membrane-bound fractions of bovine brain, based o n their ability to cleave I-125-labeled Ub-fused alpha NH-MHIS-PPEPESEEEEEH YC (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 corr esponded to UCH-L3 and UCH-L1/PGP 9.5, respectively. Intriguingly, the part iculate fraction of the brain homogenate was found to also contain strong a ctivities against I-125-Ub-PESTc, which can be solubilized by treatment wit h 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 D UBs (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 chromato graphic operations. These mDUBs differed from each other in substrate speci ficity and exhibited different characteristics from the sDUBs, revealing th at they are a new type of membrane-bound DUE. These results indicate the pr esence of divergent DUBs in mammalian brain, which may contribute to regula tion of numerous pivotal cellular functions mediated by the covalent modifi cation of Ub.