RESUMO
Zn(2+) and Cd(2+) ion exchange between transcription factor IIIA (TFIIIA) and apo-metallothionein (MT) were studied using a combination of methods including chromatography, ultrafiltration and UV spectroscopy. Under near stoichiometric conditions, apoMT was able to remove most if not all of the zinc ions from TFIIIA, whether or not the TFIIIA was bound to the 5S DNA internal control region (ICR), and concomitantly inhibit its DNA-binding activity as indicated by an electrophoretic mobility shift assay. The kinetics of the two processes were similar. The rate of the metal exchange reaction increased with the concentrations of both reactants. A second-order rate constant of 30+/-10 M(-1)s(-1) was calculated. Similar observations were made for the reaction between apoMT and Cd-substituted TFIIIA, which proceeded without observable intermediates according to a spectrophotometric analysis. A very slow metal ion exchange occurred between Cd-TFIIIA and Zn-MT, but not between Cd-MT and Zn-TFIIIA. Comparative studies on the reaction of TFIIIA with a small competing ligand, ethylenedinitrilo-tetraacetic acid (EDTA), were also conducted. Although EDTA reacts with free Zn-TFIIIA, under similar conditions it failed to compete for Zn(2+) bound as Zn-TFIIIA-ICR.
