Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates.
Ubiquitin-lysozyme conjugates have been used as substrates to identify an ATP-dependent protease from rabbit reticulocyte lysates. The enzyme, which has been partially purified by DEAE chromatography and glycerol gradient centrifugation, has an apparent molecular weight greater than 600,000 based on sedimentation and gel filtration. Whereas it degrades conjugated lysozyme molecules in the presence of ATP, the protease does not degrade free lysozyme molecules even upon addition of ubiquitin, lysozyme-ubiquitin conjugates, and ATP. Degradation of lysozyme conjugates is independent of added ubiquitin and occurs in fractions incapable of ubiquitin conjugation. Proteolysis is maximal at pH 7.8, inhibited by hemin, N-ethylmaleimide, or aurintricarboxylic acid, and proceeds with an apparent Arrhenius activation energy in the range of 27 +/- 5 kcal/mol. These properties are similar to those observed for the degradation of lysozyme conjugates in lysates indicating that the partially purified protease catalyzes the "second" ATP-utilizing reaction identified previously (Hough, R., and Rechsteiner, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 90-94; Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1619-1623; Tanaka, K., Waxman, L., and Goldberg, A. L. (1983) J. Cell Biol. 96, 1580-1585).
D049069:ATP-Dependent Proteases; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006360:Heat-Shock Proteins; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009113:Muramidase / Q000378:metabolism*; D010455:Peptides / Q000032:analysis; D011480:Protease Inhibitors; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D012697:Serine Endopeptidases; D014452:Ubiquitins / Q000378:metabolism*
D006360:Heat-Shock Proteins; D006609:High Mobility Group Proteins; D010455:Peptides; D011480:Protease Inhibitors; D014452:Ubiquitins; D001312:Aurintricarboxylic Acid; D000255:Adenosine Triphosphate; D009113:Muramidase; D010450:Endopeptidases; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; D005033:Ethylmaleimide