Binding of inorganic phosphate to the cadmium‐induced dimeric form of metallothionein from rabbit liver

Recently we have demonstrated that the exposure of monomeric Cd 7 ‐metallothionein (MT) to Cd(II) ions in potassium phosphate buffer results in the nonoxidative formation of MT dimers containing approximately two additional Cd(II) ions/monomer subunit [Palumaa, P., Mackay, E. and Vašák, M. (1992) Biochemistry 31 , 2181–2186]. In this study, we demonstrate that inorganic phosphate participates in the Cd‐induced dimerization of MT. In the absence of phosphate, Cd‐ induced oligomerization of MT still takes place, but a substantially lower apparent yield of the dimeric form and an additional peak of MT tetramers were detected in gel‐filtration experiments. Arsenate exhibits a similar effect to that of phosphate, whereas a number of other anions, i.e. F − , NO 3 − , SO 4 2− , ClO 4 − , BO 3 − , SCN − , HCOO − and CH 3 COO − had no effect on Cd‐induced oligomerization of MT. Studies on the pH dependence of MT dimerization indicate that the dianionic form of phosphate is involved in this process. Equilibrium–dialysis experiments using potassium [ 32 P]phosphate established binding of two molecules of phosphate to the dimeric MT form with a dissociation constant, K d , of 23 ± 3 μM (20 mM Tris/HCl and 0.1 M K Cl, pH 8.0 at 25°C), whereas binding of phosphate was not observed with the monomeric Cd 7 ‐MT. The nonovalent nature of phosphate binding to the Cd‐induced MT dimers has been demonstrated. The presented data provide the first evidence for the binding of a nonmetallic cellular component to MT.