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Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin‐ligated proteins by closely linked mechanisms

KANAYAMA, Hiro‐omi, TAMURA, Tomohiro, UGAI, Shinichi, KAGAWA, Susumu, TANAHASHI, Nobuyuki, YOSHIMURA, Tetsuro, TANAKA, Keiji, ICHIHARA, Akira
European journal of biochemistry 1992 v.206 no.2 pp. 567-578
adenosine triphosphate, adenosinetriphosphatase, agarose, chromatography, conjugated proteins, cytosol, electrophoresis, glycerol, humans, hydroxyapatite, kidneys, molecular weight, proteasome endopeptidase complex, protein degradation, proteinases, proteolysis, ubiquitin, ultracentrifugation
It is known that two types of high‐molecular‐mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP‐dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney, In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 × g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular‐sieve chromatography on a Biogel A‐1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density‐gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21–31 kDa and 13–15 protein components ranging in molecular mass over 35–110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I‐labeled lysozyme‐ubiquitin conjugates in an ATP‐dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP‐dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy‐dependent, ubiquitin‐mediated protein degradation.