Fujinami K, Tanahashi N, Tanaka K, Ichihara A, Cejka Z, Baumeister W, Miyawaki M, Sato T, Nakagawa H
Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University, Japan.
J Biol Chem. 1994 Oct 14;269(41):25905-10.
The 26 S proteasome complex catalyzing ATP-dependent breakdown of ubiquitin-ligated proteins was purified from spinach leaves to near homogeneity by chromatography on DEAE-cellulose, gel filtration on Biogel A-1.5, and glycerol density gradient centrifugation. The purified enzyme was shown to degrade multi-ubiquitinated, but not unmodified, lysozymes in an ATP-dependent fashion coupled with ATPase activity supplying energy for proteolysis and isopeptidase activity to generate free ubiquitin. By nondenaturing electrophoresis, the purified enzyme was separated into two distinct forms of the 26 S complex, named 26 S alpha and 26 S beta proteasomes, with different electrophoretic mobilities. The 26 S proteasome was found to consist of multiple polypeptides with molecular masses of 23-35 and 39-115 kDa, which were thought to be those of a 20 S proteasome with multicatalytic proteinase activity and an associated regulatory part with ATPase and deubiquitinating activities, respectively. The subunit multiplicity of the spinach 26 S proteasome closely resembled that of rat liver with minor differences in certain components. No sulfhydryl bond was involved in the assembly of this multicomponent polypeptide complex. Electron microscopy showed that the 26 S proteasome complex had a "caterpillar"-like shape, consisting of four central protein layers, assumed to be the 20 S proteasome, with asymmetric V-shaped layers at each end. These structural and functional characteristics of the spinach 26 S proteasome showed marked similarity to those of the mammalian 26 S proteasomes reported recently, suggesting that the 26 S proteasome is widely distributed in eukaryotic cells and is of general importance for catalyzing the soluble energy- and ubiquitin-dependent proteolytic pathway.
通过DEAE - 纤维素柱层析、Biogel A - 1.5凝胶过滤和甘油密度梯度离心，从菠菜叶中纯化出催化泛素连接蛋白ATP依赖性降解的26 S蛋白酶体复合物，纯度接近均一。纯化后的酶显示能以ATP依赖性方式降解多聚泛素化的溶菌酶，而非未修饰的溶菌酶，同时伴有为蛋白水解提供能量的ATP酶活性以及产生游离泛素的异肽酶活性。通过非变性电泳，纯化后的酶被分离为26 S复合物的两种不同形式，分别命名为26 Sα蛋白酶体和26 Sβ蛋白酶体，具有不同的电泳迁移率。发现26 S蛋白酶体由分子量为23 - 35 kDa和39 - 115 kDa的多种多肽组成，它们分别被认为是具有多催化蛋白酶活性的20 S蛋白酶体以及具有ATP酶和去泛素化活性的相关调节部分。菠菜26 S蛋白酶体的亚基多样性与大鼠肝脏的非常相似，仅某些成分存在细微差异。该多组分多肽复合物的组装不涉及巯基键。电子显微镜显示，26 S蛋白酶体复合物呈“毛虫”状，由四个中央蛋白层组成，推测为20 S蛋白酶体，两端各有一个不对称的V形层。菠菜26 S蛋白酶体的这些结构和功能特征与最近报道的哺乳动物26 S蛋白酶体显著相似，表明2 S蛋白酶体广泛分布于真核细胞中，对于催化可溶性能量和泛素依赖性蛋白水解途径具有普遍重要性。
