Interconversion of Mn²⁺-dependent and -independent protein phosphatase 2A from human erythrocytes: Role of Zn²⁺ and Fe²⁺ in protein phosphatase 2A

Human erythrocyte Mn²⁺-dependent (C'A') and -independent (CA) protein-serine/threonine phosphatase (PP) 2A are composed of 34-kDa catalytic C' and C subunits, in which the metal dependency resides, and 63-kDa regulatory A' and A subunits, respectively. Each catalytic and regulatory subunit gave the same V8- and papain-peptide maps, respectively. Stoichiometric zinc and substoichiometric iron were detected in CA but not in C'A'. The Mn²⁺-dependent protein-tyrosine phosphatase (PTP) activity of C'A' was about 70-fold higher than that of CA. Pre-incubation of CA with 25 mM NaF changed CA to a Mn²⁺-dependent form with higher PTP activity. The same NaF treatment had no effect on C'A'. Pre-incubation of C'A' with ZnCl₂, zinc-metallothionein, or FeCl₂ activated the Mn²⁺-independent PP activity, but pre-incubation with FeCl₃ did not. Ascorbate in the pre-incubation and assay mixture significantly stimulated the effect of FeCl₂. Pre-incubation of C'A' with 5 μM ZnCl₂ and 15 μM FeCl₂ in the presence of 1 mM ascorbate synergistically stimulated the Mn²⁺-independent PP activity, with concomitant suppression of the Mn²⁺ -dependent PP and PTP activities. The PP and PTP activities of CA were unaffected by the same zinc and/or iron treatment. Micromolar concentrations of vanadate strongly inhibited the Mn²⁺-dependent PP activity of C'A' but only slightly inhibited the PP activity of CA. Using the distinct effect of vanadate as an indicator, the interconversion between CA and C'A' with the above mentioned treatments was proved. These results support the notion that Mn²⁺-independent CA is a Zn²⁺- and Fe²⁺-metalloenzyme, whose apoenzyme is Mn²⁺-dependent C'A'.