Hershko A, Eytan E, Ciechanover A, Haas A L
J Biol Chem. 1982 Dec 10;257(23):13964-70.
Previous studies in a cell-free proteolytic system from reticulocytes indicated that the conjugation of ubiquitin with proteins plays a role in protein breakdown. To examine some of the physiological functions of the ubiquitin conjugation system, and immunochemical method was developed for the isolation of ubiquitin-protein conjugates from intact cells. A specific antiserum was raised against ubiquitin and purified by affinity chromatography on ubiquitin-Sepharose. When cells are labeled with tryptophan (which is missing from ubiquitin), labeled immunoreactive material isolated by the antibody is derived from the protein moiety of ubiquitin-protein conjugates. There is a marked increase in the labeling of ubiquitin-protein conjugates during the formation of abnormal proteins in reticulocytes (induced by the incorporation of amino acid analogs), suggesting that proteins with abnormal structure are more readily conjugated to ubiquitin than most normal proteins. Essentially similar, although less marked, effects of amino acid analogs were observed in Ehrlich ascites cells. When further protein synthesis was blocked with cycloheximide, ubiquitin conjugates decayed more extensively than the corresponding average labeled cellular proteins. This is consistent with the interpretation that a considerable part of ubiquitin conjugates is derived from a pool of rapidly degradable proteins.
先前在网织红细胞无细胞蛋白水解系统中的研究表明，泛素与蛋白质的结合在蛋白质降解中起作用。为了研究泛素结合系统的一些生理功能，开发了一种免疫化学方法，用于从完整细胞中分离泛素 - 蛋白质缀合物。制备了针对泛素的特异性抗血清，并通过在泛素 - 琼脂糖上的亲和色谱法进行纯化。当细胞用色氨酸（泛素中不存在）标记时，通过抗体分离的标记免疫反应性物质来自泛素 - 蛋白质缀合物的蛋白质部分。在网织红细胞中形成异常蛋白质（由氨基酸类似物掺入诱导）期间，泛素 - 蛋白质缀合物的标记显著增加，这表明结构异常的蛋白质比大多数正常蛋白质更容易与泛素结合。在艾氏腹水细胞中观察到氨基酸类似物的作用基本相似，尽管不太明显。当用环己酰亚胺阻断进一步的蛋白质合成时，泛素缀合物的降解比相应的平均标记细胞蛋白质更广泛。这与以下解释一致，即相当一部分泛素缀合物来自快速降解的蛋白质池。
