title: Partial purification and substrate specificity of a ubiquitin hydrolase from Saccharomyces cerevisiae.
abstract: A ubiquitin hydrolase that removes ubiquitin from a multi-ubiquitinated protein has been purified 600-fold from Saccharomyces cerevisiae. Four different ubiquitin-protein conjugates were assayed as substrates during the purification procedure. Enzymic activities that removed ubiquitin from ubiquitinated histone H2A, a ubiquitin-ubiquitin dimer and a ubiquitin-ribosomal fusion protein were separated during the purification from an activity that removed a single ubiquitin molecule linked by an isopeptide bond to a ubiquitinated protein. The size of the native enzyme was 160 kDa, based on its sedimentation in a sucrose gradient, and the subunit molecular mass was estimated to be 160 kDa, based on a profile of proteins eluted in different fractions by thiol-affinity chromatography. The partially purified hydrolase was not inhibited by a variety of protease inhibitors, except for thiol-blocking reagents. The natural substrate for this enzyme may be the polyubiquitin chain containing ubiquitin molecules bound to each other in isopeptide bonds, with one of them linked to a lysine residue of a protein targeted for intracellular proteolysis.
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
authors.forename: N
authors.initials: N
authors.lastname: Agell
authors.forename: C
authors.initials: C
authors.lastname: Ryan
authors.forename: M J
authors.initials: MJ
authors.lastname: Schlesinger
mesh_terms: D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D010450:Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase