Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity

Summary

The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn²⁺, was unaffected by Mg²⁺, Ca²⁻, and Ba⁺, and was markedly inhibited by Ni²⁻, Fe²⁺, Zn²⁺ and Cu²⁻. When assayed in the presence of calmodulin, many divalent metals (Ni²⁻, Zn²⁺, Pb²⁺, Cd²⁺), besides Mn²⁺, increased modestly the phosphatase activity at low concentrations (10–100 μM) and inhibited it markedly at high concentrations. Ca²⁻-calmodulin stimulated phosphatase activity was antagonized by Ni²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Pb²⁺, at low concentrations (50 μM), and by Ba²⁺, Cd²⁺ at slightly higher concentrations (> 100 μM); Mn²⁺ and Co²⁻ (50 μM to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn²⁺ (± calmodulin) and Ca²⁺ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn²⁺ led to a high activity conformation state of calcineurin that was ‘long-lived’ or ‘pseudo-irreversible’. Such Mn²⁺-activated state of calcineurin exhibited no discerbible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg²⁺ supported the intrinsic phosphatase activity, although to a lesser degree than Mn²⁺. The latter cation, compared to Mg²⁺ and Ni²⁺, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.