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.