Pittman, R.C. et al. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem. J. 212, 791−800

Biochemical Journal (Impact Factor: 4.4). 07/1983; 212(3):791-800. DOI: 10.1042/bj2120791
Source: PubMed


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.

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    • "Radio-labelled tyramine cellobiose (125I-TC) was prepared by reacting TC with Na125I (Perkin-Elmer) in Iodogen tubes (Pierce) and was then coupled covalently to heat-denatured collagen (5 mg in 500 μl borate buffer, pH 9.4) after activating the 125I-TC with the cross-linking agent cyanuric chloride, essentially as described by Pittman et al [58]. The labelled protein (125I-TC-collagen) was dialyzed against PBS using 10000 Da dialysis cassettes (Pierce Biotechnology) to remove non-coupled 125I-TC. "
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    • "This technique uses non-hydrolyzable radioactive tracers which become trapped in tissues following uptake. This allows for the measurement of catabolism of HDL by specific tissues without the release of free label into plasma (Glass et al., 1983a; Pittman et al., 1983; Glass et al., 1983b). HDL cholesterol is taken up by tissues either through HDL holo-particle internalization or selective uptake of esterified cholesterol (cholesteryl ester, CE) via scavenger receptor BI (SR-BI) (Acton et al., 1996). "
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    • "As described previously, lysozyme was labeled with radioactive iodine via TC, a label that is retained intracellularly after lysosomal degradation of the protein (Haas et al., 1993). Briefly, the synthesis of TC was performed by reductive amination of cellobiose with tyramine (Pittman et al., 1983). Cellobiose (10 mmol), 10 mmol tyramine hydrochloride, and 10 mmol propionic acid were dissolved in 40 ml of methanol. "

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