Interconversion of Mn(2+)-dependent and -independent protein phosphatase 2A from human erythrocytes: role of Zn(2+) and Fe(2+) in protein phosphatase 2A.
Human erythrocyte Mn(2+)-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' [Nishito et al. (1999) FEBS Lett. 447, 29-33]. The Mn(2+)-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 Mn(2+)-dependent form with higher PTP activity. The same NaF treatment had no effect on C'A'. Pre-incubation of C'A' with ZnCl(2), zinc-metallothionein, or FeCl(2) activated the Mn(2+)-independent PP activity, but pre-incubation with FeCl(3) did not. Ascorbate in the pre-incubation and assay mixture significantly stimulated the effect of FeCl(2). Pre-incubation of C'A' with 5 microM ZnCl(2) and 15 microM FeCl(2) in the presence of 1 mM ascorbate synergistically stimulated the Mn(2+)-independent PP activity, with concomitant suppression of the Mn(2+)-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 Mn(2+)-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 Mn(2+)-independent CA is a Zn(2+)- and Fe(2+)-metalloenzyme, whose apoenzyme is Mn(2+)-dependent C'A'.
Nishito
Y
Y
Department of Biochemistry, Hiroshima University School of Medicine, Minami-ku, Hiroshima, 734-8551, Japan.
Usui
H
H
Tanabe
O
O
Shimizu
M
M
Takeda
M
M
D000818:Animals; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D054648:Protein Phosphatase 2; D012969:Sodium Fluoride / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism*
D004791:Enzyme Inhibitors; D014638:Vanadates; D008345:Manganese; D012969:Sodium Fluoride; D007501:Iron; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D015032:Zinc