title: Stoichiometry and dynamic interaction of metal ion activators with calcineurin phosphatase.
abstract: 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 (Pallen, C. J., and Wang, J. H. (1984) J. Biol. Chem. 259, 6134-6141) was correlated with the release of the bound ions, thus suggesting that the bound ion is directly responsible for enzyme activation.
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005374:Filtration; D008345:Manganese / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis / Q000378:metabolism*; D013054:Spectrophotometry, Atomic
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008345:Manganese; D009532:Nickel; D004492:Edetic Acid; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium