The degradation of guanidinated lysozyme in reticulocyte lysate.
Egg white lysozyme, treated with O-methylisourea to convert lysine to homoarginine residues, was used as a substrate for the ATP-dependent proteolytic pathway in rabbit reticulocyte lysates. Although guanidinated lysozyme was degraded by an ATP-dependent, hemin-sensitive process, ubiquitin conjugates of this protein were present at less than 5% the level of conjugates between ubiquitin and nonguanidinated lysozyme. When lysates were chromatographed on DEAE-cellulose to produce Fractions I and II of (Hershko et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3107), ubiquitin-depleted Fraction II was capable of degrading nonguanidinated lysozyme, but the degradation of guanidinated lysozyme was markedly reduced or abolished. Glycerol-stabilized Fraction II, on the other hand, supported the degradation of both proteins in an ATP-dependent process stimulated by ubiquitin. The degradation of the two proteins differed, however, in that guanidinated lysozyme was more sensitive to competitive substrates, and higher concentrations of ubiquitin were required for its maximal proteolysis. Despite ubiquitin stimulation of guanidinated lysozyme degradation, only trace amounts of higher molecular weight species of guanidinated lysozyme attributable to ubiquitin conjugation were observed in ubiquitin-supplemented, glycerol-stabilized Fraction II even when special precautions were employed to preserve labile covalent bonds. These results indicate that covalent attachment of ubiquitin to the epsilon-amino group of substrate lysines is not mandatory for ATP-dependent proteolysis in rabbit reticulocyte lysates. The observation that ubiquitin stimulates proteolysis of guanidinated lysozyme, without extensive conjugation to it, suggests that ubiquitin may have essential functions for proteolysis other than direct marking of the protein substrate.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D006427:Hemin / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008970:Molecular Weight; D009113:Muramidase / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D014452:Ubiquitins / Q000494:pharmacology
D014452:Ubiquitins; D006427:Hemin; D000255:Adenosine Triphosphate; D009113:Muramidase