Regulation of proton release from HeLa cells by ferric reductase

Willetta Elaine Toole, Purdue University

Abstract

Diferric transferrin is known to be essential for the growth of many mammalian cells in culture. The basis for this requirement can clearly be based on the transport of ferric iron into the cell via a transferrin receptor. The iron can then be incorporated into essential cellular proteins such as myoglobin and hemoglobin. Neither the mechanism of iron uptake from transferrin nor the stimulation of cell proliferation by transferrin is completely understood. Questions still remain about the site of iron uptake and the mechanism employed to transfer the impermeable ferric iron through the plasma membrane. The discovery of a ferric reductase in the plasma membrane has introduced a possible new mechanism for iron uptake from diferric transferrin. The reductase may also provide a mechanism for growth stimulation by diferric transferrin. Transferrin may fulfill these mechanisms by serving as an electron acceptor for the transmembrane dehydrogenase and also by supplying iron for uptake from endocytotic vesicles. Reduction of iron on diferric transferrin at the plasma membrane is associated with release of protons from the cells and an increase in cytosolic NAD. Investigation of the proton release shows that it is dependent on the action of the Na+/H+ antiport in the plasma membrane which is dependent on external sodium, is accompanied by an increase in intracellular pH, and is inhibited by amiloride and amiloride analogs. Evidence that electron transport is necessary to activate the Na+/H+ antiport is developed from several approaches including the effects of antitransferrin antibodies, antitransferrin receptor monoclonal antibodies, apotransferrin, and chloroquine. Studies using antitransferrin monoclonal antibodies illustrated further the involvement of the transferrin receptor in the reduction of iron. The reduction of iron was inhibited by high concentrations of antimicrotubule drugs. The evidence presented is consistent with a ferric iron reductase in the plasma membrane which functions through the transferrin receptor. Activation of electron transport through this reductase increases the activity of the Na+/H+ antiport of the cell surface, changes the NAD/NADH ratio, and increases intracellular pH. These responses could provide a basis for control of cell proliferation.

Degree

Ph.D.

Advisors

Crane, Purdue University.

Subject Area

Biology

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