Ugai S, Tamura T, Tanahashi N, Takai S, Komi N, Chung C H, Tanaka K, Ichihara A
First Department of Surgery, School of Medicine, University of Tokushima.
J Biochem. 1993 Jun;113(6):754-68. doi: 10.1093/oxfordjournals.jbchem.a124116.
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 of 21-31 and 35-110 kDa, respectively. The subunit multiplicity of the rat 26S proteasome closely resembled that of the human counterpart, showing only minor species-specific differences in certain components. The assembly of this multi-component complex was found not to involve a sulfhydryl bond. Electrophoretic peptide mapping with lysyl-endopeptidase indicated the non-identity of the multiple subunits of the 26S proteasome. From these structural and functional characteristics, the 26S proteasome, which is widely distributed in mammals, is suggested to be a new type of multi-molecular complex catalyzing the soluble energy- and ubiquitin-dependent proteolytic pathway.
通过一种基于先前报道程序改进的方法，从大鼠肝脏中纯化出一种沉降系数约为26S的ATP/泛素依赖性蛋白酶体复合物，纯度接近均一。不仅在纯化制剂中，而且在粗提物中都发现了两种电泳性质不同的26S复合物形式，分别命名为26Sα和26Sβ，它们的亚基组成非常相似，这表明26S蛋白酶体以两种异构体形式存在。研究表明，26S蛋白酶体以ATP依赖性方式降解多泛素化的溶菌酶，而非未修饰的溶菌酶，具有为蛋白水解提供能量的ATP酶活性，并含有能依赖Mg2+/ATP产生游离泛素的异肽酶活性。26S蛋白酶体还分别催化三种在切割位点带有碱性、中性和酸性氨基酸的荧光肽的非ATP依赖性水解。这些肽也是20S蛋白酶体的良好底物，但它们被游离的20S蛋白酶体及其在26S复合物中的组装形式降解的情况明显不同，这表明两种形式的蛋白酶体在功能上存在差异。电泳和免疫化学分析表明，大的26S复合物大致由两种不同结构组成：一种具有多催化蛋白酶功能的核心20S蛋白酶体和一个可能具有调节作用的相关部分。这两种结构均由分子量分别为21 - 31 kDa和35 - 110 kDa的多种多肽组成。大鼠26S蛋白酶体的亚基多样性与人类的非常相似，仅在某些成分上存在微小的物种特异性差异。发现这种多组分复合物的组装不涉及巯基键。用赖氨酰内肽酶进行的电泳肽图谱分析表明26S蛋白酶体的多个亚基各不相同。从这些结构和功能特征来看，广泛分布于哺乳动物中的26S蛋白酶体被认为是一种新型的多分子复合物，催化可溶性的能量和泛素依赖性蛋白水解途径。
