Control of zinc transfer between thionein, metallothionein, and zinc proteins

Abstract

Metallothionein (MT), despite its high metal binding constant (KZn = 3.2 x 10(13) M-1 at pH 7.4), can transfer zinc to the apoforms of zinc enzymes that have inherently lower stability constants. To gain insight into this paradox, we have studied zinc transfer between zinc enzymes and MT. Zinc can be transferred in both directions-i.e., from the enzymes to thionein (the apoform of MT) and from MT to the apoenzymes. Agents that mediate or enhance zinc transfer have been identified that provide kinetic pathways in either direction. MT does not transfer all of its seven zinc atoms to an apoenzyme, but apparently contains at least one that is more prone to transfer than the others. Modification of thiol ligands in MT zinc clusters increases the total number of zinc ions released and, hence, the extent of transfer. Aside from disulfide reagents, we show that selenium compounds are potential cellular enhancers of zinc transfer from MT to apoenzymes. Zinc transfer from zinc enzymes to thionein, on the other hand, is mediated by zinc-chelating agents such as Tris buffer, citrate, or glutathione. Redox agents are asymmetrically involved in both directions of zinc transfer. For example, reduced glutathione mediates zinc transfer from enzymes to thionein, whereas glutathione disulfide oxidizes MT with enhanced release of zinc and transfer of zinc to apoenzymes. Therefore, the cellular redox state as well as the concentration of other biological chelating agents might well determine the direction of zinc transfer and ultimately affect zinc distribution.

Figure 1

Concentration dependence of reconstitution of apo-AP with Zn₇MT-2 (⧫) and Cd₅Zn₂MT-2 (▪). Apo-AP, 0.5 μM, was incubated with various concentrations of Zn₇MT-2 and Cd₅Zn₂MT-2 in 10 mM Tris⋅HCl, pH 8.0. Aliquots were taken after 2 hr and assayed spectrophotometrically for enzymatic activity.

Figure 2

Reconstitution of apo-AP with Zn₇MT-2 in the presence of oxidizing agents. Apo-AP, 0.5 μM, was incubated with Zn₇MT-2, 0.29 μM, and various concentrations of DTNB (⧫) or GSSG (▪) in 10 mM Tris⋅HCl, pH 8.0. Aliquots were taken after 2 hr and assayed spectrophotometrically for enzymatic activity. AP activity of 100% corresponds to reactivated apo-AP in the absence of oxidizing agents under otherwise identical conditions.

Figure 3

Concentration dependence of reconstitution of apo-CPA with Zn₇MT-2. Apo-CPA, 2 μM, was incubated with various concentrations of Zn₇MT-2 in 20 mM Hepes/100 mM NaCl, pH 7.5. Measurements were taken after 30 min.

Figure 4

Reaction of MT-2 with PAR in the presence of selenite or selenocystamine. MT-2, 0.5 μM, was incubated with PAR, 100 μM, in the absence (•) and presence of 50 μM selenocystamine (▪) or 50 μM sodium selenite (⧫).

Figure 5

Time dependence of the inactivation of AP by T. AP, 0.5 μM, was incubated with T, 1 μM, in 1 M Tris⋅HCl, pH 8.0. Aliquots were taken at defined time intervals and assayed spectrophotometrically for enzymatic activity.

Figure 6

Effect of Zn(II) on the reaction of T with DTNB. T, 0.5 μM, was incubated with various concentrations of zinc sulfate in 20 mM Hepes, pH 7.5, for 30 s, and DTNB was added to a final concentration of 50 μM. Relative reactivity of thiols was determined from measurements taken after 30 s.