A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo.
A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo.
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一种放射性碘标记的细胞内捕获配体，用于确定体内血浆蛋白降解的位点。
DOI：
10.1042/bj2120791
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Attie,AD
中科院分区：
文献类型：
--
作者：
Pittman,RC;Carew,TE;Glass,CK;Green,SR;TaylorJr,CA;Attie,AD
我们最近开发了一种测定血浆蛋白在体内降解的组织位点的通用方法，该方法利用共价附着的放射性蔗糖。在蛋白质的降解过程中，蔗糖作为蛋白质降解的累积标记留在细胞中。这里描述的方法依赖于相同的原理，但使用了一种纤维素二糖和酪胺的加合物，该加合物被放射到高比放射性，然后共价地附着在蛋白质上。使用放射性碘化配体使该方法的灵敏度提高至少100倍，并允许简化组织分析。用放射性碘化配体衍生的蛋白质在体外和体内都被认为是欠激活的蛋白质。在衍生化低密度脂蛋白的降解中，培养成纤维细胞在24小时内的渗漏率仅为5%。同样，将标记的蛋白质注射到大鼠和家兔中，在所有情况下，在注射后24小时，尿中排泄的标记物少于总标记分解代谢产物的10%。在大鼠和家兔注射有标记的asialofetuin或载脂蛋白A1后两次检测标签的组织含量，发现标签在初始摄取和分解代谢后没有在组织间重新分布；通过出现在肠道内容物和粪便中的标签，定量地解释了肝脏的显著泄漏。设计了一种简单的双标记方法，用于校正被困血浆、血管外空间和细胞内的完整蛋白。使用这种方法，就不需要对组织样品进行分馏。
We recently developed a general method for determining tissue sites of degradation of plasma proteins in vivo that made use of covalently attached radioactive sucrose. On degradation of the protein, the sucrose remained trapped in the cells as a cumulative marker of protein degradation. The method described here depends on the same principles, but uses an adduct of cellobiose and tyramine that is radioiodinated to high specific radioactivity and then covalently attached to protein. Use of the radioiodinated ligand increases the sensitivity of the method at least 100-fold and allows simplified tissue analysis. Proteins derivatized with the radioiodinated ligand were recognized as underivatized proteins both in vitro and in vivo. On degradation of derivatized low-density lipoprotein, the rate of leakage from cultured fibroblasts was only 5% during 24 h. Similarly, on injection of labelled proteins into rats and rabbits, urinary excretion of the label was in all cases less than 10% of total labelled catabolic products recovered 24 h after injection. Examination of the tissue contents of label at two times after injection of labelled asialofetuin or apolipoprotein A1 in rats, and asialotransferrin in rabbits showed that the label did not detectably redistribute between tissues after initial uptake and catabolism; a significant leakage from liver was quantitatively accounted for by label appearing in gut contents and faeces. A simple double-label method was devised to provide a correction for intact protein in trapped plasma, the extravascular spaces, and within cells. By using this method it becomes unnecessary to fractionate tissue samples.