Stoichiometry and dynamic interaction of metal ion activators with calcineurin phosphatase

Calcineurin, a calmodulin-regulated phosphatase, is composed of two distinct subunits (A and B) and requires certain metal ions for activity. The binding of the two most potent activators, Ni²⁺ and Mn²⁺, to calcineurin and its subunits has been studied. Incubation of the protein with ⁶³Ni²⁺ (or ⁵⁴Mn²⁺) followed by gel filtration to separate free and protein-bound ions indicated that calcineurin could maximally bind 2 mol/mol of Ni²⁺ or Mn²⁺. While isolated A subunit also bound 2 mol/mol of Ni²⁺, no Mn²⁺ binding was demonstrated for either isolated A or B subunit. When bindings were monitored by nitrocellulose filter assay, only 1 mol/mol bound Ni²⁺ or Mn²⁺ was detected, suggesting that the two Ni²⁺ (or Mn²⁺) binding sites had different relative affinities and that only metal ions bound at the higher affinity sites were detected by the filter assay. Preincubation of calcineurin with Mn²⁺ (or Ni²⁺) decreased the filter assay-measured Ni²⁺ (or Mn²⁺) binding by only 30%. Preincubation of the protein with Zn²⁺ decreased the filter assay-measured Ni²⁺ or Mn²⁺ binding by 90 or 17%, respectively. The results suggest that the higher affinity sites are a Ni²⁺-specific site and a distinct Mn²⁺-specific site. Preincubation of calcineurin with Mn²⁺ (or Ni²⁺) decreased the gel filtration-determined Ni²⁺ (or Mn²⁺) binding from 2 to 1 mol/mol suggesting that calcineurin also contains a site which binds either metal ion. The time course of Ni²⁺ (or Mn²⁺) binding was correlated with that of the enzyme activation, and the extent of deactivation of the Ni²⁺-activated calcineurin by EDTA or by incubation with Ca²⁺ and calmodulin was correlated with the release of the bound ions, thus suggesting that the bound ion is directly responsible for enzyme activation.