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
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
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DOI：
10.1111/j.1432-1033.1992.tb16961.x
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发表时间：
1992-06-01
期刊：
影响因子：
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通讯作者：
ICHIHARA, A
中科院分区：
文献类型：
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作者：
KANAYAMA, HO;TAMURA, T;ICHIHARA, A
已知，哺乳动物细胞的胞浆中存在两种类型的高相对分子质量的蛋白酶复合体：一种是20S潜伏的多催化酶，称为蛋白酶体；另一种是大的蛋白分解复合体，其表观沉降系数为26S，催化与泛素结合的蛋白质依赖于ATP的分解。在这项工作中，我们首先证明了激活潜伏的蛋白酶体需要低浓度的十二烷基硫酸钠，而26S复合体在没有十二烷基硫酸钠的情况下降解了蛋白酶体的底物。此外，在2 mM的ATP和20%的甘油存在下，26S复合体得到了极大的稳定。基于这些特性，我们接下来设计了一种从人肾中分离纯化26S蛋白水解物的新方法。在这个过程中，用105000×g的超速离心法从细胞质提取液中沉淀5h的蛋白水解物，并在Biogel A-1.5M柱上用分子筛色谱从20S蛋白酶体中清楚地分离出大的26S复合体。用羟基磷灰石和Q琼脂糖柱层析，然后用甘油密度梯度离心法纯化26S酶，纯化至表观均一。电泳和免疫化学分析表明，纯化的人26S复合体由多个蛋白酶体亚基组成，分子量在21-31 kDa之间，13-15个蛋白质组分在35-110 kDa以上，与蛋白酶体直接相关。纯化的26S蛋白水解物以依赖于ATP的方式降解I-125标记的溶菌酶-泛素结合物。26S酶也表现出较高的ATPase活性，并与该复合体发生了相互作用。钒酸根和氯化高铁血红素不仅强烈地抑制了ATP的裂解。但也依赖于ATP对泛素连接蛋白的分解，这表明26S复合体通过紧密相连的机制对ATP和泛素连接蛋白进行水解。这些发现表明，26S复合体由一个具有蛋白分解功能的蛋白酶体和多个其他成分组成，其中包括一个调节能量依赖的泛素介导的蛋白质降解的ATPase。
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 x 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 I-125-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 ubiquitin-ligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitin-ligated 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.