Significance of extracellular zinc‐binding ligands in the uptake of zinc by human fibroblasts

Abstract

Extracellular zinc (Zn)-binding ligands were investigated as vehicles for uptake of Zn by human fibroblasts. The uptake of α-macroglobulin, a major serum Zn-binding protein proposed to have a function in Zn transport, was less than 1/200 that of the Zn uptake rate. The fibroblast growth medium, BME with 10% FBS, contains several Zn-binding ligands. These were separated into components of MW greater than 30,000 and components of MW less than 30,000 using an Amicon microconcentrator. Cells accumulated Zn from both fractions; however, there was more uptake from the filtrate (MW 30,000), containing ligands with low affinity for Zn, hence with greater free Zn concentration. Zn uptake from a number of ligands with a range of affinities for Zn was examined and found to be inversely proportional to the K_a value for the ligands and therefore proportional to the free Zn concentration. When histidine and desferrioxamine, two structurally different Zn-binding ligands were compared, analysis of the concentration curves of calculated free Zn against Zn uptake gave similar V_max and Km values (S.E.M.) (±S.E.M.) of 373 ± 6 pmol/μg DNA'h and 0.08 ± 0.004 μM for histidine, and 349 ± 10 pmol/μg DNA/h and 0.06 ± 0.008 μM for DFO, suggesting that the same transport mechanism was operating in both systems. We conclude that no specific ligands are essential for transport of Zn into fibroblasts, but that ‘free’ Zn is acquired by the cell.