Direct metal analyses of Mn 2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn 2+-independent one

Abstract

A Mn 2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C′ subunit and a 63 kDa regulatory A′ subunit, was purified from human erythrocyte cytosol. C′ and A′ produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn 2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C′A and CA′ revealed that the metal dependency resided in C′ and not in A′. In CA, 0.87±0.12 mol zinc and 0.35±0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C′A′. Pre-incubation of C′ with ZnCl 2 and FeCl 2, but not FeCl 3, synergistically stimulated the Mn 2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn 2+- and Fe 2+-metalloenzyme and that C′ is the apoenzyme.