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.
A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo
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R C Pittman, T E Carew, C K Glass, S R Green, C A Taylor, A D Attie; A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem J 15 June 1983; 212 (3): 791–800. doi: https://doi.org/10.1042/bj2120791
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