A role for ATPase in the mechanisms of ATP-dependent Ca and phosphate deposition by isolated rachitic matrix vesicles

Abstract

Physiological levels of ATP support mammalian matrix vesicle-mediated calcium deposition although the released Pi from ATP did not cause the [Ca] × [PO 4 ] ion product to exceed the threshold for spontaneous precipitation of calcium phosphate (Hsu, Biochem. Biophys. Acta 1116, 227–233, 1992). The aim of this report was to see if a specific ATPase is involved in ATP-stimulated Ca 2+ and phosphate deposition. Since ATP can be non-specifically cleaved by alkaline phosphatase (ALP), thus obscuring a specific ATPase activity, phosphatidylinositol specific phospholipase C (PI-PLC) which selectively removes ALP from isolated matrix vesicle membranes and l -tetramisole (LT), a specific ALP inhibitor were used to allow studies on specific membrane associated ATPase. The specific ATPase activity and ATP-dependent Ca and P deposition were then tested for their correlation. [ γ - 32 P]ATP hydrolytic activity of PI-PLC-released alkaline phosphatase (ALP) was not stimulated by Ca 2+ whereas membrane-associated ATPase was stimulated dose-dependently by addition of Ca 2+ or Mg 2+ . LT at 1 mM completely inhibited solubilized ALP activity whereas it only partially inhibited membrane ATPase activity. The apparent affinity of membrane ATPase, [K 0.5 ], for Ca 2+ , Mg 2+ , and ATP was 10, 20, and 49 μ M, respectively. Membrane perturbing agents such as 1% Triton X-100 or 5% butanol abolished either Ca 2+ or Mg 2+ stimulation of membrane associated ATPase. The ALP-depleted matrix vesicles with ATPase activity was able to deposit both Ca and Pi when ATP was present in calcifying media. In contrast, PI-PLC released ALP failed to deposit Ca or Pi. ATP-dependent Ca and P depositing activity by matrix vesicles was completely inhibited when the membrane was ruptured by 10% butanol. The high affinity of ATPase for Ca 2+ and ATP, insensitivity of ATPase and ATP-dependent Ca and P depositing activities to LT or PI-PLC treatments, and membrane dependence of stimulation of ATPase by Ca 2+ suggest the presence of a specific ATPase responsible for a part of [ γ - 32 P]ATP hydrolysis and involved in ATP-dependent deposition of Ca and phosphate by matrix vesicles.