Raffinose, a Novel Radioactive Label for Determining Organ Sites of Catabolism of Proteins in the Circulation Jonathon Van Zile
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University of South Carolina Scholar Commons Faculty Publications Chemistry and Biochemistry, Department of 9-6-1978 [3H]‑Raffinose, a Novel Radioactive Label for Determining Organ Sites of Catabolism of Proteins in the Circulation Jonathon Van Zile Lee A. Henderson John W. Baynes University of South Carolina - Columbia, [email protected] Suzanne R. Thorpe Follow this and additional works at: https://scholarcommons.sc.edu/chem_facpub Part of the Chemistry Commons Publication Info 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. This Article is brought to you by the Chemistry and Biochemistry, Department of at Scholar Commons. It has been accepted for inclusion in Faculty Publications by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. : [3H]Raffinose, a novel radioactive label for determining organ sites of catabolism of proteins in the circulation. J Van Zile, L A Henderson, J W Baynes and S R Thorpe J. Biol. Chem. 1979, 254:3547-3553. Downloaded from Access the most updated version of this article at http://www.jbc.org/ http://www.jbc.org/content/254/9/3547.citation Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites . Alerts: at USC SCHOOL OF MEDICINE on September 30, 2014 • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/254/9/3547.citation.full.html#ref-list-1 THE JOURNAL OF BVXOG~CA~ CHEMI~TRV Vol. 254, No. 9, Issue of May 10. pp. 3547-3553, 1979 Printedin U.S.A. [3HjRaffinose, a Novel Radioactive Label for Determining Organ Sites of Catabolism of Proteins in the Circulation* (Received for publication, September 6, 1978) Jonathan Van Zile, Lee A. Henderson,$ John W. Baynes, and Suzanne R. Thorpe8 From the Department of Chemistry, College of Science and Mathematics, and School of Medicine, University of South Carolina, C&umbia, South Carol&a 29268 The primary tissue sites of catabolism of plasma covalently coupled to plasma proteins should be a use- proteins with long circulating half-lives are unknown. ful radioactive tracer for detecting the tissue and cel- It has been difficult to identify these sites because lular sites of catabolism of long lived circulating pro- plasma proteins are delivered to tissues at relatively teins. Downloaded from slow rates but are rapidly degraded intracellularly within lysosomes. Therefore, a tracer attached to pro- tein is lost from the site of uptake before an amount There is extensive information concerning the sites of syn- sufficient for quantitation can accumulate. We hypoth- thesis and kinetics of turnover of plasma proteins; however, esized that sucrose (Glupal-2 /3Fruf) would be a useful there is little firm evidence concerning their mechanisms of label to circumvent this difficulty because of the stabil- http://www.jbc.org/ ity of sucrose in lysosomes; and thus, sucrose should clearance from the circulation or their tissue sites of degra- dation (2-4). Perhaps the greatest technical difficulty in ob- remain in tissue long after the protein to which it was attached had been degraded to products released from taining this information has been the absence of a suitable the lysosome. [G-3HJRaffinose (RAF, Galpal- Glupcwl- radioactive label with which to monitor the sites of catabolism 2flFruf) was selected as the vehicle for attaching su- of proteins with the long circulating half-lives characteristic of plasma proteins. In general, the rate of intracellular degra- crose to protein. [31XjRAF was converted to the C-6 at USC SCHOOL OF MEDICINE on September 30, 2014 aldehydogalactose form with galactose oxidase and dation of plasma proteins within lysosomes far exceeds the then covalently coupled to protein by reductive ami- rate of clearance of these long lived proteins. Thus, if plasma nation using NaBH3CN. proteins are labeled with conventional labels, such as ? or [‘H]RAF was coupled first to two relatively long lived radioactive amino acids, the tracer does not accumulate at the plasma proteins, bovine serum albumin and fetuin. The site of catabolism, but, rather, as the protein is degraded to half-lives of these proteins in the rat circulation (&,* = monomeric products which exit from the lysosome, the label -24 h) was unchanged, suggesting that RAF did not is lost to cellular or extracellular metabolic pools, or both. In alter the normal mechanisms of protein clearance. contrast, the sites of catabolism of proteins with short circu- When attached to short lived proteins with known sites lating half-lives are readily identified since their rates of of catabolism, such as asialofetuin, RNase B, and heat- clearance are relatively rapid, compared to the rate of lyso- denatured albumin, neither the tissue nor cellular sites somal digestion. It has been possible, therefore, to identify the of uptake of the proteins were altered. Thus, 13H]RAF- liver as the primary site for catabolism of [“H,%u]asialocer- asialofetuin was recovered almost exclusively (>90%) uloplasmin (5) and lz51-RNase B (6) injected in rats, and in the liver parenchymal cell fraction, while both bovine /3-glucuronidase injected in mice (7), all proteins which [JHJRAF-labeled RNase B and heat-denatured albumin exhibit half-lives of 15 min or less. were recovered primarily (>85%) in nonparenchymal In order to circumvent the difficulties inherent in determin- cells. In addition, the RAF label was observed to reside ing the in. uiuo fate of long lived plasma proteins, a tracer is stably (tl,2 > 100 h) in the liver following degradation of the carrier protein; in contrast, radioactivity from “‘I- needed which will 1) not affect mechanisms of protein clear- labeled asialofetuin or RNase B was rapidly (tl,z < 30 ance and catabolism, 2) be resistant to degradation by lyso- min) lost from liver. Radioactivity from 13H]RAF-pro- somal hydrolases, and 3) accumulate in lysosomes at the site teins was recovered in a lysosomally enriched subcel- of the labeled protein’s degradation. We hypothesized that lular fraction in liver and consisted of a low molecular sucrose (Glcpcyl-ZPFru,) covalently attached to protein weight species (-llOO), containing both glucose and through its glucosyl moiety might fulfill these criteria for the fructose in a ratio similar to that in the original pro- following reasons. First, because of the stable equilibration of tein. The results of these studies establish that 13H]RAF inulin (a fructan) and rafiinose (RAF, Galpcul-GGlcpal- 2/3Frun into plasma and extravascular spaces in uivo (8), it * This work was supported by United States Public Health Service seemed unlikely that the terminal fructose residues on a Research Grant AM19971 and National Institutes of Health Biomed- ical Research Support Grant 5S07RR07160. A preliminary report of protein would serve as recognition markers for a carbohy- this work has appeared (1). The costs of publication of this article drate-dependent clearance process analogous to those already were defrayed in part by the payment of page charges. This article described for galactose (9), fucose (lo), mannose (11, 12), and must therefore be hereby marked “aduertisement” in accordance N-acetylglucosamine (13,14) terminal glycoproteins. Second, with 18 USC. Section 1734 solely to indicate this fact. because of the absence of significant levels of mammalian * This work has been submitted, in part, in partial fulfillment of lysosomal sucrase activity (15), it seemed unlikely that hy- the requirements for a Master of Science degree in Chemistry from the University of South Carolina. drolysis of the sucrose moiety would occur either before or 5 To whom all correspondence should be addressed at the Depart- after degradation of the protein. Finally, since sucrose does ment of Chemistry, University of South Carolina, Columbia, S. C. not readily diffuse from lysosomes (15), it or its amino acid 29208. conjugate should accumulate at this subcellular site of protein 3547 3548 Determination of Sites of Catabolism of Circulating Proteins catabolism, despite the fact that other components of the lation fluid (Beckman Bio-Solv EP). Weighed portions (-1 g) of tissue protein would exit from the lysosome following degradation. from ““I-protein injection experiments were counted directly in a As a vehicle for the attachment of sucrose to protein, we Beckman Gamma 4000 gamma counter. Isolation of parenchymal and non-parenchymal cells of liver by collagenase digestion and differen- chose the trisaccharide [G-3H]raffinose as our radioactive tial centrifugation was carried out as previously described (6). Sub- starting material. Oxidation of the C-6 hydroxyl of the galac- cellular fractionation of liver by differential centrifugation into nu- tose residue, using galactose oxidase (16), provided an alde- clear, mitochondrial/lysosomal, and supernatant fractions was per- hyde function which could be readily coupled to protein, formed according to Gregoriadis and Sourkes (22) using P-N-acetyl- under mild conditions, using NaBH3CN (17). We present hexosaminidase activity (23) as a lysosomal marker enzyme. below the results obtained in vitro for the coupling of RAF’ In order to characterize products resulting from the in uiuo degra- dation of [gH]RAF-labeled proteins, livers were taken at various times to several proteins by this method. We also present our results postinjection of heat-denatured albumin or asialofetuin; the livers from studies on the comparative fates of 12?- and [“HIRAF- were homogenized in 4 volumes of H20, frozen, and thawed four to labeled proteins in viuo, as a test of our hypothesis that RAF five times and then centrifuged at 12,000 x g for 30 min.