Purification and initial characterization of ubiquitin from the higher plant, Avena sativa.

摘要: Ubiquitin is a highly conserved, 76-amino acid polypeptide recently demonstrated to be involved in ATP-dependent protein degradation mammalian cells. From immunoblot analyses with anti-human-ubiquitin antibodies we have detected the presence of free ubiquitin green leaves, etiolated shoots, and dry seeds higher plant, oats (Avena sativa L.). We also find that crude oat extracts contain protease(s) rapidly degrade both human (t1/2 approximately 10 min at 27 degrees C). This proteolysis apparently cleaves carboxyl-terminal glycine dipeptide results inactivation molecule respect ligation but does not affect its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using homogenization conditions preclude this (low pH addition protease inhibitor p-chloromercuribenzoate) immunoblotting as an assay for protein, procedure purification from shoots was developed. Characterization purified by absorption spectra, electrophoresis, isoelectric focusing, radioimmunoassay antibodies, kinetic using activating enzyme isolated rabbit liver indicates remarkably similar form. Small differences between proteins been observed amino compositional indicating two forms are totally homologous. Immunoblotting has revealed high molecular weight recognized anti-ubiquitin represent ubiquitin-protein conjugates formed vivo. Taken together, these data provide evidence plants ubiquitin-dependent proteolytic pathway mechanistically identical present animals.