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Ascorbate Enhances Copper Transport from Ceruloplasmin into Human K562 Cells¹

Department of Biochemistry and Biophysics and the Texas Agricultural Experiment Station, Texas A&M University, College Station, TX 77843

Copper uptake from human ceruloplasmin (Cp) into cells of a human erythroleukemic cell line, K562, was investigated. The interaction between ascorbic acid and the copper atoms in ceruloplasmin was a focal point of the study. Nondenatured ⁶⁷Cu-labeled ceruloplasmin (⁶⁷Cu-Cp) was prepared by an ascorbate-catalyzed exchange of Cp with ⁶⁷CuCl₂ in vitro. The complex was stable, even in the presence of 1.0 mM ascorbate. Adding K562 cells and incubating at 37°C resulted in an immediate transfer of ⁶⁷Cu from ceruloplasmin to the cells. At 37°C the copper accumulated by the K562 cells resisted dissociation by mild acid washing. The rate of transfer of ⁶⁷Cu was proportional to the Cp concentration in the medium. Ascorbate (100 µM) enhanced the uptake of ⁶⁷Cu at least fourfold. D-Isoascorbate worked as well as L-ascorbate, suggesting that the reducing potential of the vitamin (or its isomer) was important in the uptake of copper. Approximately 20% of the ⁶⁷Cu absorbed into the cytosol was precipitable with antibodies to Cu-Zn superoxide dismutase (Cu-Zn SOD). Ascorbate, however, did not enhance the incorporation of radioactivity into Cu-Zn SOD, suggesting that copper may not be the only rate-limiting factor in the synthesis of this enzyme in K562 cells. The possible relevance of these observations to vitamin C deficiency is discussed.

KEY WORDS: • copper transport • copper delivery • ceruloplasmin • ascorbic acid • vitamin C • erythroleukemic cells • superoxide dismutase

¹ Supported by United States Public Health Service grant DK35920 from the National Institutes of Health and Hatch Project H-6621 of the Texas Agricultural Experiment Station.

² To whom reprint requests should be addressed.

Manuscript received 12 September 1988. Revision accepted 10 January 1989.