The primary tissue sites of catabolism of plasma proteins with long circulating half-lives are unknown. It has been difficult to identify these sites because plasma proteins are delivered to tissues at relatively slow rates but are rapidly degraded intracellularly within lysosomes. Therefore, a tracer attached to protein is lost from the site of uptake before an amount sufficient for quantitation can accumulate. We hypothesized that sucrose (Glupal-2 /3Fruf) would be a useful label to circumvent this difficulty because of the stability of sucrose in lysosomes; and thus, sucrose should remain in tissue long after the protein to which it was attached had been degraded to products released from the lysosome. [G-3HJRaffinose (RAF, Galpal- Glupcwl- 2flFruf) was selected as the vehicle for attaching sucrose to protein. [31XjRAF was converted to the C-6 aldehydogalactose form with galactose oxidase and then covalently coupled to protein by reductive amination using NaBH3CN. [‘H]RAF was coupled first to two relatively long lived plasma proteins, bovine serum albumin and fetuin. The half-lives of these proteins in the rat circulation (&,* = -24 h) was unchanged, suggesting that RAF did not alter the normal mechanisms of protein clearance. When attached to short lived proteins with known sites of catabolism, such as asialofetuin, RNase B, and heatdenatured albumin, neither the tissue nor cellular sites of uptake of the proteins were altered. Thus, 13H]RAFasialofetuin was recovered almost exclusively (>90%) in the liver parenchymal cell fraction, while both [JHJRAF-labeled RNase B and heat-denatured albumin were recovered primarily (>85%) in nonparenchymal cells. In addition, the RAF label was observed to reside stably (tl,2 > 100 h) in the liver following degradation of the carrier protein; in contrast, radioactivity from “‘Ilabeled asialofetuin or RNase B was rapidly (tl,z < 30 min) lost from liver. Radioactivity from 13H]RAF-proteins was recovered in a lysosomally enriched subcellular fraction in liver and consisted of a low molecular weight species (-llOO), containing both glucose and fructose in a ratio similar to that in the original protein. The results of these studies establish that 13H]RAF useful radioactive tracer for detecting the tissue and cellular sites of catabolism of long lived circulating proteins.
Published in Journal of Biological Chemistry, Volume 254, Issue 9, 1978, pages 3547-3553.
This research was originally published in the Journal of Biological Chemistry. Zile J, Henderson LA, Baynes JW, Thorpe SR. [3H] Raffinose, a Novel Radioactive Label for Determining Organ Sites of Catabolism of Proteins in the Circulation. Journal of Biological Chemistry. 1978; 254: 3547-3553. © the American Society for Biochemistry and Molecular Biology.