Regulation of smooth muscle phosphatase-II by divalent cations

Abstract

Smooth Muscle Phosphatases II (SMP-II) which has been purified from turkey gizzards and previously classified as protein phosphatase 2C, is inactive in the absence of divalent cations. Study of the activation of SMP-II by Mg2+ and Mn2+ revealed differences in the modes of activation by these cations. The maximal activation elicited by Mg2+ is 1.5-2.5-fold higher than the maximal Mn2+ activation. However, the latter is achieved at a lower concentration than the maximal Mg2(+)-activation. Furthermore, at low cation concentrations (less than or equal to 2 mM), the Mn2(+)-activated activity is higher than the Mg2(+)-activated activity. In the presence of both cations, the effect of Mn2+ predominates suggesting that the affinity of the enzyme for Mn2+ is greater than for Mg2+. In contrast to Mg2+ and Mn2+, Ca2+ does not activate SMP-II but it was observed to antagonize the effects of Mg2+ and Mn2+. Ca2+ acts as a competitive inhibitor of Mg2+. However, the inhibitory effect at high Ca2+ concentrations is not completely reversed by increasing the Mg2+ concentration. Mn2+ activation is also inhibited by Ca2+ but to a lesser extent. Ca2+ cannot completely inhibit Mn2(+)-activation suggesting that SMP-II has greater affinity for Mn2+ than for Ca2+. The finding that Ca2+ inhibits the activation of SMP-II raises the possibility that Ca2+ may be a regulator of SMP-II in vivo.

Kinetic Analysis of Human Serine/Threonine Protein Phosphatase 2Cα

“…Metal Dependence-The nature of the metal cation has been suggested to be important for regulating the activity of PP2C (18). To explore which divalent cations are effective in activating PP2C, k cat and k cat /K m values were determined for several divalent cations at saturating pNPP concentrations.…”

The Catalytic Mechanisms of Binuclear Metallohydrolases

“…The biologically relevant metal ions are unclear, but human PP2C is activated most efficiently by both Fe(II) and Mn(II) and to some extent by Mg(II), while Cu(II), Ni(II), and Zn(II) tend to inhibit enzyme activity. 139 For the recently detected Pst/Ppp from M. tuberculosis, metal ion analysis detected only the presence of manganese; no significant quantities of iron or zinc were found. 117 Furthermore, in assays, Mg(II), Ca(II), Ba(II), Zn(II), and Li(I) were not able to substitute Mn(II) in vitro, even at concentrations as high as 4 mM.…”

Purification and characterization of calponin phosphatase from smooth muscle. Effect of dephosphorylation on calponin function

“…Phosphatase activity was quantified by liquidscintillation counting of the remaining 32P-labelled protein as previously described (Walsh et al, 1983 ATPase activities were measured as previously described (Ikebe & Hartshorne, 1985) Other procedures Thiophosphorylated LC20-Sepharose was prepared as previously described (Pato & Kerc, 1990). Protein concentrations were determined by the Coomassie Brilliant Blue dye-binding assay (Spector, 1978) using dye reagent purchased from Pierce Chemical Co. (Rockford, IL, U.S.A.), or by spectrophotometric measurements using the following molar absorption coefficients: calmodulin e1l; 1.9 (Klee, 1977), myosin el'/ 4.5 (Okamoto & Sekine, 1978), tropomyosin el2 2.9 (Eisenberg & Kielley, 1974), caldesmon elj/ 3.3 (Graceffa et al, 1988) and calponin e1/ 11.3 (Winder & Walsh, 1990a).…”

“…The purified calponin phosphatase was stored at -20°C after further dialysis against buffer B containing 20 mM-NaCl. In one case the pooled activity from the AOA column was used to prepare the catalytic subunit of the phosphatase as previously described (Pato & Kerc, 1990).…”