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

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Abstract
Recently we have demonstrated that the exposure of monomeric Cd7-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., Mackey, E. and Vasák, 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-, NO3-, SO4(2-), ClO4-, BO3-, SCN-, HCOO- and CH3COO- 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 [32P]phosphate established binding of two molecules of phosphate to the dimeric MT form with a dissociation constant, Kd, of 23 +/- 3 microM (20 mM Tris/HCl and 0.1 M KCl, pH 8.0 at 25 degrees C), whereas binding of phosphate was not observed with the monomeric Cd7-MT. The noncovalent 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.