Information about copper metabolism is extremely limited. There is, however, an important interaction between copper and ascorbate that must be clarified, one critical to our general understanding of copper transport and uptake by cells. The present proposal aims at characterizing this valuable link. The studies are built on data supporting the concept L-dihydroascobate, the vitamin, is capable under conditions permitted by host cells to mobilize cooper from ceruloplasmin, the major serum copper protein. Ascorbate is hypothesized to be either a catalyst in the dissociation (direct effect) or a cofactor for an enzyme system within the cell or on the cell membrane (indirect effect). Either way, ascorbate is postulated to transfer reducing equivalents to ceruloplasmin which ultimately frees the tightly bound copper from the protein and makes it available for cells and copper-requiring enzymes in the cytosol. K562 cells of human origin will be used in the first phase of the work. The cells will be treated with hemin (20 uM) to induce maximum binding of ceruloplasmin. 67Cu-labeled ceruloplasmin will be prepared by procedures that do not denature the protein. The criteria for deciding cooper transport will be based on quantitative measurements of 67Cu transferred to an acid- resistant compartment of the cells or to a specific cytosolic enzyme. CuZn superoxide dismutase (CuZnSOD). The latter will be identified by immunoprecipitation. The influence of ascorbate and other reducing sources including ascorbate derivatives iron, cysteine, will be evaluated in the transfer. In a second phase of the study, the turnover of 67Cu-ceruloplasmin will be evaluated in guinea pigs raised on diets lacking ascorbate or fed high levels of the vitamin. Control and experimental animals will be compared for whole body retention of an administered dose of 67Cu. Short- term studies will evaluate the rate of turnover of copper in ceruloplasmin as a function of the ascorbate administered. The kinetics of copper transfer from ceruloplasmin to cytosolic CuZnSOD in heart tissue, blood cells and liver will evaluate the physiological; significance of the event. In all, these studies are designed to determine whether there are cause-effect relationships between ascorbate deficiency and/or dietary excess and copper utilization. The cellular studies should determine the site where the vitamin and copper share a common pathway.
| Davidson, L A; McOrmond, S L; Harris, E D (1994) Characterization of a particulate pathway for copper in K562 cells. Biochim Biophys Acta 1221:1-6 |
| Harris, E D (1993) The transport of copper. Prog Clin Biol Res 380:163-79 |
| Harris, E D (1992) Regulation of antioxidant enzymes. FASEB J 6:2675-83 |
| Harris, E D (1992) Copper as a cofactor and regulator of copper,zinc superoxide dismutase. J Nutr 122:636-40 |
| Harris, E D; Percival, S S (1991) A role for ascorbic acid in copper transport. Am J Clin Nutr 54:1193S-1197S |
| Harris, E D (1991) Copper transport: an overview. Proc Soc Exp Biol Med 196:130-40 |
