The interactions of zinc, nickel, and cadmium with Xenopus transcription factor IIIA, assessed by equilibrium dialysis

Abstract

Transcription factor IIIA (TFIIIA) was isolated from Xenopus ovary and treated with 1,10-phenanthroline to remove zinc. The interactions of apoTFIIIA with Zn 2+, Ni 2+, and Cd 2+ were studied by equilibrium dialysis under anaerobic conditions (pH 7.0, 25°C), using 65ZnCl 2, 62NiCl 2, and 109CdCl 2, as the radioligands. The data for binding of Zn 2+, Ni 2+, and Cd 2+ to apoTFIIIA were best-fitted by a model with two classes of binding sites. For Zn 2+, the apparent dissociation constants ( K dl Zn and K d 2 Zn) for the high- and low-affinity sites were 1.0 × 10 −8 and 2.6 × 10 −5 M; the apparent binding capacities of the two classes were 0.8 ± 0.5 and 9.6 ± 0.3 g-atoms of Zn/mol; the Hill coefficient was 1.18, consistent with positive cooperativity of Zn-binding sites. For Ni 2+, the apparent K dl Ni and K d 2 Ni values were 2.3 × 10 −5 and 5.2 × 10 −4 M; the apparent binding capacities were 2.3 ± 0.6 and 8.6 ± 0.6 g-atoms of Ni/mol; the Hill coefficient was 1.20 consistent with positive cooperativity of Ni-binding sites. For Cd 2+, the apparent K dl Cd and K d 2 Cd values were 2.8 × 10 −6 and 1.6 × 10 −4 M; the apparent binding capacities were 0.9 ± 0.3 and 2.4 ± 0.5 g-atoms of Cd/mol; the Hill coefficient was 0.53, consistent with negative cooperativity or heterogeneity of Cd-binding sites. This study has the following significance: First, it helps to resolve a controversy about the zinc content of purified TFIIIA. Second, it shows that the K dl Zn of apoTFIIIA is less than the reported K d Zn of thionein, consistent with the hypothesis that thionein modulates gene expression by competing with TFIIIA and other Zn-finger proteins for intracellular Zn 2+ stores. Third, it confirms previous indirect evidence that the affinity of apoTFIIIA for Zn 2+ is much greater than for Cd 2+, and that the affinity for Cd 2+ is greater than for Ni 2+.