Enzymatic characterization of the matrix vesicle alkaline phosphatase isolated from bovine fetal epiphyseal cartilage

Summary

Purified matrix vesicle alkaline phosphatase from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and PP_i. Optimal activities forp-nitrophenyl phosphate, ATP, and PP_i are found at pH 10.5, 10.0, and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations.p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasing substrate concentration. Heat inactivation studies indicate that both phosphorohydrolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg²⁺ and Hg²⁺ ions inhibit thep-nitrophenyl phosphatase activity at pH 10.5 while Mn²⁺ ions show no effect. P_i, levamisole, CN⁻, Zn²⁺, Ca²⁺ ions, and L-phenylalanine are reversible inhibitors of the phosphomonoesterase activity. P_i is a linear noncompetitive inhibitor with a K_i of 8.0 mM. Levamisole and L-phenylalanine are uncompetitive inhibitors with inhibition constants of 0.02 and 39.4 mM, respectively. Ca²⁺ ions inhibit noncompetitively with a K_i of 9.3 mM. Zn²⁺ ion is a potent noncompetitive inhibitor with an inhibition constant of 0.026 mM. The enzyme is inhibited irreversibly by Be²⁺ ion, EDTA, EGTA, ethane-l-hydroxydiphosphonate, dichloromethanediphosphonate, L-cysteine, and N-ethylmaleimide. NaCl, KCl, and Na₂SO₄ at 0.5–1.0 M inhibit the enzyme.

At pH 8.5, the cleavage of PP_i by the matrix vesicle enzyme is inhibited by Mg²⁺ and Ca²⁺ ions at concentrations greater than 0.5 mM. Mg²⁺ ions in the range of 0.1–4 mM stimulate the matrix vesicle ATPase whereas higher concentrations produce inhibition. Ca²⁺ ion does not affect the ATPase activity between 0.1 and 10 mM at either pH 7.5 or 10.0. With 5′-AMP as substrate, the 3.5-fold decrease in the maximum velocity of the matrix vesicle relative to the chondrocyte enzyme may be accounted for in terms of a small excess enthalpy of activation (1160 cal/mole) partially compensated by an increased entropy of activation.