Purification and Characterization of the 26S Proteasome Complex Catalyzing ATP-Dependent Breakdown of Ubiquitin-Ligated Prot from Rat Liver1

作者：Shin‐ichi Ugai, Tomohiro Tamura, Nobuyuki Tanahashi, Shigeham Takai, N Komi, Chin Ha Chung, Keiji Tanaka, Akira Ichihara · 发表于：The Journal of Biochemistry · 年份：1993 · DOI：10.1093/oxfordjournals.jbchem.a124116 · 被引用次数：88 · 研究领域：Ubiquitin and proteasome pathways、Peptidase Inhibition and Analysis、Genetics and Neurodevelopmental Disorders

An ATP/ubiquitin-dependent proteasome complex with an apparent sedimentation coefficient of 26S was purified from rat liver to near homogeneity by an improved method based on procedures reported previously. Two electrophoretically distinct forms of the 26S complex, named 26S alpha and 26S beta, with very similar subunit compositions were found not only in purified preparations but also in crude extracts, indicating that the 26S proteasome is present as two isoforms. The 26S proteasome was shown to degrade multi-ubiquitinated, but not unmodified, lysozymes in an ATP-dependent fashion, to have ATPase activity supplying energy for proteolysis, and to contain isopeptidase activity to generate free ubiquitin Mg2+/ATP-dependently. The 26S proteasome also catalyzed the ATP-independent hydrolyses of three types of fluorogenic peptides with basic, neutral, and acidic amino acids at their cleavage sites, respectively. These peptides are also good substrates for the 20S proteasome, but their degradation by the free 20S proteasome and by its assembled form in the 26S complex differ markedly, suggesting a functional difference between the two forms of proteasomes. Electrophoretic and immunochemical analyses showed that the large 26S complex was composed grossly of two different structures: a core 20S proteasome with multicatalytic proteinase functions and an associated part possibly with a regulatory role. These two structures both consisted of multiple polypeptides with molecular masses ...