The inactivation of ubiquitin accounts for the inability to demonstrate ATP, ubiquitin-dependent proteolysis in liver extracts.
The low molecular weight polypeptide required for energy-dependent proteolysis, ubiquitin, is rapidly inactivated by 100,000 X g supernatants of rabbit liver extracts. Ubiquitin inactivation results from limited proteolysis by an endogenous contaminating lysosomal thiol protease having trypsin-like specificity. Evidence for this includes a pH optimum of 5.0 for the first order constant of ubiquitin inactivation and observation that inactivation is inhibited by EDTA, o-phenanthroline, iodoacetamide, p-chloromercuribenzoic acid, phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethyl ketone, leupeptin, soybean trypsin inhibitor, and aprotinin. Metals stimulate but are not required for ubiquitin inactivation with the effect apparently mediated by a low molecular weight heat-labile component of crude extracts. When this heat-labile component is removed by gel exclusion chromatography a number of metals inhibit ubiquitin inactivation. In the presence of excess dithiothreitol, inhibition is relatively specific for Zn(II). Inhibition by Zn(II) is specifically overcome competitively by Cd(II) or by a concentration of ubiquitin in excess of Zn(II). The responsible cathepsin possesses a molecular mass of 35 kDa by gel exclusion chromatography and shows marked thermal lability at neutral pH but stability at acid pH. Proteolytic inactivation of ubiquitin results from limited cleavage of the carboxyl-terminal glycine dipeptide required for isopeptide bond formation and is supported by data on isoelectric point changes on subsequent digestion with carboxypeptidase B and by direct amino acid analysis. When the responsible cathepsin is inactivated, liver extracts display ATP,ubiquitin-dependent proteolysis that cannot be ascribed to contaminating erythrocytes. Thus the previous inability to demonstrate energy-dependent proteolysis in liver extracts is accounted for by the artifactual inactivation of ubiquitin.
Haas
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Murphy
K E
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Bright
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PM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004492:Edetic Acid / Q000494:pharmacology; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*; D011817:Rabbits; D014452:Ubiquitins / Q000378:metabolism*
D006609:High Mobility Group Proteins; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D010447:Peptide Hydrolases