Multiple (α-NH-ubiquitin)Protein Endoproteases in Cells

• Published in: The Journal of biological chemistry 1989-06, Vol.264 (18), p.10637-10642
• Main Authors: Jonnalagadda, S, Butt, T R, Monia, B P, Mirabelli, C K, Gotlib, L, Ecker, D J, Crooke, S T
• Format: Article
• Language: English
• Subjects: ( alpha -NH-ubiquitin)protein proteinase; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chromatography, Ion Exchange; Endopeptidases - blood; Endopeptidases - isolation & purification; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Kinetics; Ligases - metabolism; Molecular Weight; rabbits; reticulocytes; Reticulocytes - enzymology; Substrate Specificity; Ubiquitin-Activating Enzymes; Ubiquitin-Protein Ligases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)81669-6

Ubiquitin is encoded as a variable, spacerless repeat of the gene terminating with an additional amino acid or as a gene coding for a single ubiquitin with a carboxyl-terminal extension of 52 to 80 amino acids. We report the identification and partial purification of enzymes that specifically hydrolyze the peptide bond between ubiquitin-ubiquitin conjugate (Ub-Ubase) or ubiquitin fusion proteins (Ub-Xase). The Ub-Ubase was separated from the Ub-Xase by dye-ligand-Sepharose chromatography. The Ub-Xase was purified further by affinity chromatography on ubiquitin-Sepharose. The fidelity of the endoprotease reaction was assessed by measuring the ability of the released ubiquitin to be activated by ubiquitin-activating enzyme (E1) which requires intact ubiquitin and by sequence analysis of the released carboxyl extension protein with 52 amino acids after endoproteolysis of human ubiquitin with 52-amino acid carboxyl extension. The failure of both Ub-Ubase and Ub-Xase to cleave a mutant ubiquitin-Gly-76→Ala-metallothionein showed that the endoproteases distinguish Gly-X from an Ala-X peptide bonds.