Purification and initial characterization of ubiquitin from the higher plant, Avena sativa.
Ubiquitin is a highly conserved, 76-amino acid polypeptide recently demonstrated to be involved in ATP-dependent protein degradation in mammalian cells. From immunoblot analyses with anti-human-ubiquitin antibodies we have detected the presence of free ubiquitin in green leaves, etiolated shoots, and dry seeds of the higher plant, oats (Avena sativa L.). We also find that crude oat extracts contain protease(s) that rapidly degrade both oat and human ubiquitin (t1/2 approximately 10 min at 27 degrees C). This proteolysis apparently cleaves ubiquitin at the carboxyl-terminal glycine dipeptide and results in inactivation of the molecule with respect to ligation but does not affect its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using homogenization conditions that preclude this proteolysis (low pH and the addition of the protease inhibitor p-chloromercuribenzoate) and immunoblotting as an assay for the protein, a procedure for the purification of ubiquitin from etiolated oat shoots was developed. Characterization of purified oat ubiquitin by absorption spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, radioimmunoassay with anti-human-ubiquitin antibodies, and kinetic analyses using the ubiquitin activating enzyme isolated from rabbit liver indicates that this protein is remarkably similar to the mammalian form. Small differences between the oat and human proteins have been observed by amino acid compositional analyses indicating that the two forms are not totally homologous. Immunoblotting of crude oat extracts has revealed the presence of high molecular weight proteins recognized by anti-ubiquitin antibodies that represent ubiquitin-protein conjugates formed in vivo. Taken together, these data provide evidence that higher plants contain a ubiquitin-dependent proteolytic pathway that is mechanistically identical to that present in animals.
D000596:Amino Acids / Q000032:analysis; D002523:Edible Grain / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D010944:Plants / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D014452:Ubiquitins / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D006609:High Mobility Group Proteins; D011480:Protease Inhibitors; D014452:Ubiquitins; D010447:Peptide Hydrolases