Molecular Structure and Functional Propeerties of a High Molecular Weight-Multiprotease Complex

从人、大鼠、鸡肝脏、非洲爪蟾卵巢和酵母等真核生物中纯化的蛋白酶体（大型多蛋白酶复合物）具有相似的活性，如潜伏蛋白酶和多肽酶活性。所有蛋白酶体均表现出非常相似的理化性质，沉降系数为20S，分子量为800kDa。电子显微镜显示，它们也都具有相似的明确的对称形态，并且似乎是带有小孔的环状颗粒。因此，来自不同来源的蛋白酶体在大体结构上惊人地相似，但它们可以通过Ouchterlony双扩散分析和免疫印迹分析来区分，使用兔体内培养的针对每种酶的抗体。在二维电泳中，这些蛋白酶体被分离为15 ~ 20个特征组分，分子量在22 ~ 33 KDa之间，等电点在4 ~ 9之间，其亚基多样性表现出物种特异性差异。通过筛选大鼠肝脏cDNA文库，从重组cDNA克隆中确定了大鼠肝脏蛋白酶体最大组分的核苷酸序列。该亚基是单个基因的未修饰产物，这表明蛋白酶体的多个亚基可能分别由不同的基因编码。因此，蛋白酶体被认为是由不相同的多个亚基组成的不寻常的酶复合物。这项工作表明，蛋白酶体具有相似的分子结构，但在亚基多样性和免疫反应性方面存在差异，广泛分布于从人到酵母的各种真核生物中。这种无处不在的分布暗示了由这些蛋白酶体催化的蛋白质水解的普遍重要性。

Proteasomes (large multiprotease complexes) purified from various eukaryotic sources, namely human, rat and chicken livers, Xenopus laevis ovary and yeast showed similar activities, such as latent proteinase and multiple peptidase activites. All the proteasomes showed very similar physicochemical properties, such as a sedimentation coefficient of 20S and molecular weight of 800kDa. Electron microscopy showed that they all also had similar well-defined symmetrical morphology and appeared to be ring-shaped particles with a small hole. Thus, proteasomes from various sources were strikingly similar in gross structure, but they were distiguishable by Ouchterlony double diffusion analysis and immuno-blot analysis using antibodies raised in rabbits against each enzyme. On two dimensional electrophoresis, these proteasomes separated into 15-20 characteristic components with molecular weights of 22 KDa to 33 KDa and isoelectric points of 4 to 9 and their subunit multiplicity showed species-specific differences. The nucleotide sequence of the largest component of the rat liver proteasomes was determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library. This subunit is the unmodified product of a single gene, suggesting that the multiple subunits of proteasomes are likely each to be coded by a different gene. Thus, proteasomes are thought to be unusual enzyme complexes consisting of non-identical multiple subunits. This work shows that proteasomes with a similar molecular organization, but differences in subunit multiplicity and immunological reactivity are widely distributed in various eukaryotic organisms ranging from man to yeast. This ubiquitous distribution implies the general importance of proteolysis catalyzed by these proteasomes.