Interprotein metal exchange between transcription factor IIIa and apo-metallothionein

Abstract

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.

Zinc binding ligands and cellular zinc trafficking: Apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1

“…Thus, the cellular integrity of Zn 3 -Sp1 in the presence of apo-MT and GSH might be due to its existence as Zn 3 -Sp1-DNA complexes. The protection afforded Zn 3 -Sp1 through binding to a specific DNA sequence recalls a similar result with transcription factor IIIA and suggests that Zn-finger transcription factor-cognate DNA adducts might generally be kinetically inert to reaction with apo-MT [38]. The fact that the order of addition of reactants changes the outcome of the reactions points to kinetic factors as the basis for the lack of reactivity of Zn 3 -Sp1-(GC-1) with apo-MT and GSH.…”

“…Earlier papers have addressed the question whether apo-MT might compete for Cd 2+ that becomes bound to proteins in place of Zn 2+ . Both Cd-TFIIIA and Cd-carbonic anhydrase readily transfer Cd 2+ to apo-MT [38,52]. Indeed, the latter reaction occurs much more rapidly than the ligand substitution reaction between Cd-CA and EDTA.…”

Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes

“…Ligand substitution reactions involving a range of ligands, including ZI, PAR, and H 2 KTSM 2 in this work, and apo-metallothionein and EDTA in a companion paper, show that Zn 2+ in TFIIIA is kinetically as well as thermodynamically accessible to competing ligands over short time periods when the free protein participates in the reactions (Table 2) [47]. In contrast, whereas ZI reacted with free and ICR-bound protein nearly equally well, PAR was much less effective in its reaction with the protein-DNA adduct (Figs.…”

“…5(a) and (b)). Similarly, whereas apo-metallothionein reacts with either form, EDTA is singularly unreactive with the adduct [47]. Thus, the binding of the protein to a bulky DNA partner can foreclose avenues of reaction that exist in free Zn-TFIIIA.…”

“…Ligand substitution reactions were carried out to assess the reactivity and stability of the Zn-fingers in TFIIIA and Zn-F3. PAR, a ligand employed in a previous study of TFIIIA, ZI, and H 2 KTSM 2 were used in these experiments [46]; the reaction with EDTA is described in a companion study [47].…”