title: Proteolysis in heat-stressed HeLa cells. Stabilization of ubiquitin correlates with the loss of proline endopeptidase.
abstract: When intact HeLa cells were incubated at 45 degrees C, there was progressive inactivation of proline endopeptidase. Rapid loss of the enzyme did not occur in extracts maintained at 45 degrees C. Since Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no decrease in the immunoreactive 70-kDa proline endopeptidase band, its in vivo disappearance apparently results from irreversible denaturation or modification. Loss of proline endopeptidase activity was paralleled by reduced degradation of injected ubiquitin and bovine serum albumin. In contrast, proteolysis of injected lysozyme or pancreatic trypsin inhibitor was barely affected. Electrophoretic analysis of ubiquitin or bovine serum albumin retrieved from heated HeLa cells showed that the injected proteins were intact. Thus, the presence of proline endopeptidase appears to be required for initial cleavage of these two substrates, but it has not been shown that the enzyme is directly responsible. Selective stabilization of a subset of the injected proteins does, however, demonstrate the existence of distinct proteolytic pathways in HeLa cytosol.
authors.affiliation: Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
authors.forename: G
authors.initials: G
authors.lastname: Pratt
authors.forename: R
authors.initials: R
authors.lastname: Hough
authors.forename: M
authors.initials: M
authors.lastname: Rechsteiner
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D006367:HeLa Cells / Q000201:enzymology; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D000085822:Prolyl Oligopeptidases; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D013816:Thermodynamics; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D011480:Protease Inhibitors; D014452:Ubiquitins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C000705287:PREPL protein, human; D000085822:Prolyl Oligopeptidases