Histidine stimulated trace element uptake into human erythrocytes, HEL cells and HEL total RNA injected Xenopus laevis oocytes

Oakley, Fiona (2000) Histidine stimulated trace element uptake into human erythrocytes, HEL cells and HEL total RNA injected Xenopus laevis oocytes. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Zinc uptake experiments were conducted in human erythrocytes in the presence of L & D histidine, together with chelating agents to maintain a low free ionic zinc concentration. Under these conditions histidine stimulated zinc uptake into human erythrocytes in a dose dependent and stereospecific manner. Inhibition of radiolabelled zinc uptake in human erythrocytes could be achieved by the addition of 0-500 μM unlabelled divalent, but not trivalent, metal ions in the presence of both 5 and 40mM L-histidine. The histidine stimulated zinc uptake corresponded with the calculated concentration of a metal bis histidine complex as opposed to metal mono histidine complex or the free ionic metal concentration. When inhibition curves for a range of divalent metals were compared, it was found that the calculated metal bis-histidine complex concentrations corresponding to 50% inhibition of ⁶⁵Zn uptake ("apparent K_i") were very similar except in the case of manganese, where the K_i was 10 fold lower. To determine whether a Mn(his)₂ complex is competing with a Zn(his)₂ complex inhibition experiments were conducted using radiolabelled manganese. 0-500μM unlabelled manganese inhibits 40mM histidine stimulated radiolabelled manganese uptake in human erythrocytes, suggesting that the Mn(his)₂ complex competes with a Zn(his)₂ complex for transport. To investigate the transporter at the molecular level the nucleated erythroid cell line HEL.92.1.7 was used. To discover if a similar transporter was expressed in the cell line experiments were conducted to investigate the effect of L & D histidine on zinc uptake in the HEL cells in the presence of chelating agents. L histidine stimulated zinc uptake into HEL cells in a dose dependent manner whereas with the D-histidine enantiomer no stimulation of uptake was observed.