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

Abstract

Purified matrix vesicle alkaline phosphatase from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and PP~. Optimal activities for p-nitrophenyl phosphate, ATP, and PP~ 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. Mg'-'* and Hg"* ions inhibit the p-nitrophenyl phosphatase activity at pH 10.5 while Mn 2. ions show no effect. Pi, levamisole, CN-, Zn 2 § Ca ~ § ions, and L-phenylalanine are reversible inhibitors of the phosphomonoesterase activity. P~ is a linear noncompetitive inhibitor with a K~ of 8.0 mM. Levamisole and L-phenylalanine are uncompetitive inhibitors with inhibition constants of 0.02 and 39.4 mM, respectively. Ca "~ ions inhibit noncompetitively with a K~ of 9.3 raM. Zn 2+ ion is a potent noncompetitive inhibitor with an inhibition constant of 0.026 mM. The enzyme is inhibited irreversibly by Be ~ ion, EDTA, EGTA, ethane-l-hydroxydiphosphonate, dichloromelhanediphosphohate, L-cysteine. and N-ethylmaleimide. NaCI, KCI. and NaeSO4 at 0.5-1.0 M inhibit the enzyme.At pH 8.5, the cleavage of PP~ by the matrix vesicle enzyme is inhibited by Mg '+ and Ca'-'~ ions at concentrations greater than 0.5 raM. Mg 2. ions in the range of 0.1-4 mM stimulate the matrix vesicle ATPase whereas higher concentrations produce inhibition. Ca'-' ~ ion does not affect the ATPase activity between 0.1 and 10 mM at either pH 7.5 or 10.0, Send ~ffl)t'int reque.~ts to Robert P. Carx} at the above address. ~Present address:

Ecto 5′-Nucleotidase and Nonspecific Alkaline Phosphatase

“…The relatively high efficiency of ecto 5Ј-NT suggests that this enzyme would play a major role in the regulation of adenosine-mediated epithelial functions. On the other hand, all APs dephosphorylate not only AMP but also ADP and ATP (33). Under pathological conditions, trauma generates quantities of extracellular nucleotides that may cause damage to the epithelium.…”

“…Two types of ectonucleotidases could be involved in the metabolism of extracellular AMP: ecto 5Ј-NT (32) and APs (33). Their activity at the surface of human bronchial epithelial cells was detected with concanavalin A (48) and ␤-glycerophosphate (49), respectively.…”

“…32) and alkaline phosphatases (APs, EC 3.1.3.1; Ref. 33). Whereas ecto 5Ј-NT specifically dephosphorylates nucleoside monophosphates (AMP 3 adenosine (32)), APs will metabolize a spectrum of substrates, including 5Ј-nucleotides (ATP 3 ADP 3 AMP 3 adenosine), pyrophosphate, and p-nitrophenyl phosphate (33).…”

“…33). Whereas ecto 5Ј-NT specifically dephosphorylates nucleoside monophosphates (AMP 3 adenosine (32)), APs will metabolize a spectrum of substrates, including 5Ј-nucleotides (ATP 3 ADP 3 AMP 3 adenosine), pyrophosphate, and p-nitrophenyl phosphate (33). The AP family contains four ectoenzymes: intestinal AP (I AP), tissue nonspecific AP detected in several organs including liver, bone, and kidney (NS AP), placental AP (PLA AP), and germ-cell AP (G AP) reported in testis and malignant tumors (34,35).…”

Adenosine in fibrosis

“…Generation of adenosine in the extracellular milieu relies on one of two main sources: intracellular adenosine that is transported out of the cell, and the dephosphoryla-tion of AMP by the enzyme 5′-nucleotidase (CD73) or alkaline phosphatase, which can in turn be dephosphoryl-ated from adenosine triphosphate (ATP) or adenosine diphosphate (ADP) by ectonucleoside triphosphate di-phosphohydrolase 1 (CD39) or alkaline phosphatase [65–67]. Thus, deletion of CD73 or CD39 eliminates one of the major routes of adenosine formation, and gene deletion in mice provides a model of low tissue adenosine.…”