ISSN:
1432-0827
Keywords:
Bovine fetal epiphyseal cartilage
;
Matrix vesicles
;
Alkaline phosphatase
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Medicine
,
Physics
Notes:
Summary Purified matrix vesicle alkaline phosphatase from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and PPi. Optimal activities forp-nitrophenyl phosphate, ATP, and PPi 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. Pi, levamisole, CN−, Zn2+, Ca2+ ions, and L-phenylalanine are reversible inhibitors of the phosphomonoesterase activity. Pi is a linear noncompetitive inhibitor with a Ki 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 Ki 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 PPi 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF02408631
Permalink
