Effects of Zinc on Human Skeletal Alkaline Phosphatase Activity In Vitro
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Inorganic phosphate (Pi) can regulate the level of skeletal alkaline phosphatase (ALP) activity in human osteoblast-like cells by stabilizing the enzyme (without affecting transcription, ALP release from the cell surface, or the amount of ALP protein). These observations suggest that Pi determines the level of ALP activity by modulating a process of irreversible inactivation. The current studies were intended to examine the hypothesis that this inactivation of ALP activity is caused by the dissociation of an active center Zn and that Pi inhibits that dissociation. Initial studies showed that Zn, like Pi, could increase ALP specific activity in human osteosarcoma SaOS-2 cells in a time- and dose-dependent manner (e.g., a 50% increase at 0.2 μmol/liter Zn, P < 0.005). This effect was specific for Zn (i.e., no similar effect was seen with Ca, Fe, Co, Mg, Mn, or Cu), but not for SaOS-2 cells. Zn also increased ALP specific activity in (human osteosarcoma) MG-63 cells and in cells derived from normal human vertebrae (P < 0.001 for each). The effect of Zn to increase ALP activity was not associated with parallel increases in total protein synthesis, collagen production, or tartrate-resistant acid phosphatase activity (no change in any of these indices), net IGF-2 synthesis (a Zn-dependent decrease, P < 0.005), or PTH-dependent synthesis of cAMP (a biphasic increase, P < 0.02). Kinetic studies of Pi and Zn as co-effectors of ALP activity showed that Zn was a mixed-type effector with respect to Pi, whereas Pi was competitive with respect to Zn. Mechanistic studies showed that (1) Zn reversed the effect of Pi withdrawal to decrease ALP activity, but not by reactivating inactive ALP protein (the process required protein synthesis, without increases in ALP mRNA or the level of ALP immunoreactive protein); (2) Zn increased the half-life of ALP activity in intact cells and after a partial purification; and (3) Pi inhibited the process of ALP inactivation by EDTA (which chelates active center Zn). All these findings are consistent with the general hypothesis that Pi increases the half-life of skeletal ALP by preventing the dissociation of active center Zn and with a mechanistic model of skeletal ALP activity in which active center Zn participates in Pi-ester binding and/or hydrolysis.
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