Multiple (alpha-NH-ubiquitin)protein endoproteases in cells.
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.
Jonnalagadda
S
S
Department of Molecular Pharmacology, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406.
Butt
T R
TR
Monia
B P
BP
Mirabelli
C K
CK
Gotlib
L
L
Ecker
D J
DJ
Crooke
S T
ST
D000818:Animals; D002852:Chromatography, Ion Exchange; D010450:Endopeptidases / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D008025:Ligases / Q000378:metabolism; D008970:Molecular Weight; D012156:Reticulocytes / Q000201:enzymology*; D013379:Substrate Specificity; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes