The role of human liver ferritin (HuFE) in GA-67 localization

Previous work has shown that horse spleen ferritin (HoFE) has a high affinity for Ga-67 and adenosinetriphosphate (ATP) stimulates the transfer of nuclide from human transferrin (TF) and lactoferrin (LF) to HoFE which suggested an important intracellular Ga-67-sequestering role for FE. Since species variation could be great, HoFE was isolated and purified. Similar experiments were performed and results compared. A 2-chamber dialysis system, ion exchange or gel chromatography distinguished between TF, LF, HoFE or non-protein bound Ga-67. The binding of Ga-67 to HuFE(3 μM) was about 60% at 50 hours which was comparable to the HuFE results. At 30 μM HuFe the rate and amount of binding was increased to 85% at 5 hours. The transfer of Ga-67 from LF and TF to HuFE in the presence and absence of 1 mM ATP was qualitatively similar to results obtained with HuFE. Little nuclide was translocated in the promoter-free case while ATP stimulated transfer. However, quantitatively the transfers for LF→HuFE were slower and for TF→HuFe somewhat faster. An increase in the ATP concentration from 0.1-1.0 mM in the TF experiment increased the initial transfer rate (2.5-fold), % transferred to HuFE at 20 hours (1.4-fold) and decreased the difference % bound at 6 hours (33% vs - 18%). As the concentration was increased from 1 to 5 mM, the values of all parameters plateaued. In addition, ATP specifically acted on the TF-Ga-67 complex to increase non-protein bound activity even at 0.1 mM(P<.001). While between .1-1 mM ATP did not form a complex with Ga-67. These results provide additional evidence for the important role of ferritin as a Ga-67-sequestering agent and show that variation in HuFe and ATP concentrations could modulate cellular uptake. Also, this work implies ATP has a specific TF binding site and when bound reduces the affinity of TF for Ga-67