Olsnes S, Stirpe F, Sandvig K, Pihl A
J Biol Chem. 1982 Nov 25;257(22):13263-70.
A toxic protein, viscumin, was isolated from extracts of mistletoe by affinity chromatography on acid-treated Sepharose 4B. Viscumin was selectively bound to the column and could be eluted with lactose. It migrated in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate corresponding to Mr = 60,000. In addition, two bands migrating corresponding to Mr = 29,000 and 32,000 were found. After treatment with 2-mercaptoethanol, only 2 bands (Mr = 29,000 and 34,000) were found. Apparently, viscumin consists of two chains which, in some of the molecules, are disulfide-linked. Protection experiments with antiserum against viscumin indicated that the major part of the cytotoxic activity in mistletoe extracts is due to viscumin. Gel filtration experiments on Sephacryl 200 indicated that, at low concentrations, viscumin occurs as a monomer and at higher concentrations as a dimer. Viscumin was found to inhibit protein synthesis in cell-free systems. When the two constituent peptide chains of viscumin were eluted from polyacrylamide gels and tested for ability to inhibit cell-free protein synthesis, this property was found to be associated with the fastest migrating chain, here denoted the A chain. The heavier chain was denoted the B chain. The A chain was found to inhibit protein synthesis by inactivating the ribosomes catalytically. Reconstitution experiments with isolated ribosomal subunits from untreated and A chain-treated ribosomes showed that the 60 S ribosomal subunit was selectively inactivated.
通过在酸处理的琼脂糖4B上进行亲和色谱，从槲寄生提取物中分离出一种有毒蛋白质——相思豆毒素。相思豆毒素选择性地结合到柱上，可用乳糖洗脱。在十二烷基硫酸钠存在下，它在聚丙烯酰胺凝胶电泳中迁移，对应分子量为60,000。此外，还发现了两条迁移带，对应分子量为29,000和32,000。用2-巯基乙醇处理后，仅发现两条带（分子量为29,000和34,000）。显然，相思豆毒素由两条链组成，在某些分子中，这两条链通过二硫键相连。用抗相思豆毒素抗血清进行的保护实验表明，槲寄生提取物中的细胞毒性活性主要归因于相思豆毒素。在Sephacryl 200上进行的凝胶过滤实验表明，在低浓度下，相思豆毒素以单体形式存在，在高浓度下以二聚体形式存在。发现相思豆毒素可抑制无细胞系统中的蛋白质合成。当从聚丙烯酰胺凝胶中洗脱相思豆毒素的两条组成肽链，并测试其抑制无细胞蛋白质合成的能力时，发现这种特性与迁移最快的链有关，此处称为A链。较重的链称为B链。发现A链通过催化使核糖体失活来抑制蛋白质合成。用未处理的核糖体亚基和A链处理的核糖体进行的重组实验表明，60S核糖体亚基被选择性失活。
