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A soluble form of an alkaline phosphatase obtained from rat osseous plates was purified 204-fold with a yield of 24.3%. The purified enzyme showed a single protein band of Mr 80,000 on SDS-PAGE and an apparent molecular weight of 163,000 by gel filtration on Sephacryl S-300 suggesting a dimeric structure for the soluble enzyme. The specific activity of the enzyme at pH 9.4 in the presence of 2 mM MgCl2 was 19,027 U/mg and the hydrolysis of p-nitrophenyl phosphate (K0.5 = 92 microM) showed positive cooperativity (n = 1.5). The purified enzyme showed a broad substrate specificity, however, ATP, bis(p-nitrophenyl) phosphate and pyrophosphate were among the less hydrolyzed substrates assayed. Surprisingly the enzyme was not stimulated by cobalt and manganese ions, in contrast with a 20-25% stimulation observed for magnesium and calcium ions. Zinc ions exerted a strong inhibition on p-nitrophenylphosphatase activity of the enzyme. This paper provides a simple experimental procedure for the isolation of a soluble form of alkaline phosphatase which is induced by demineralized bone matrix during endochondral ossification.

1. Matrix-induced alkaline phosphatase prepared from rat osseous plate was solubilized with polidocanol and purified on a Sephacryl S-300 column. 2. Purified solubilized alkaline phosphatase has a molecular weight of ca 115,000 and bind one magnesium and two zinc ions. At least 110 detergent molecules are bound to each enzyme molecule. 3. Solubilization and purification procedures did not destroy the ability of the enzyme to hydrolyze adenosine-5'-triphosphate, p-nitrophenylphosphate, pyrophosphate and bis p-nitrophenylphosphate. 4. Magnesium, manganese and cobalt ions are stimulators of PNPPase activity of solubilized enzyme whereas calcium and zinc ions are inhibitors.

1. The inhibition of matrix-induced alkaline phosphatase by zinc ions is due to the displacement of magnesium ions from its binding site. 2. Binding of magnesium ions to alkaline phosphatase induces conformational changes which activate the enzyme. 3. Binding of zinc ions to alkaline phosphatase induces conformational changes which impair the catalytic action of the enzyme. 4. The inhibition of the enzyme by zinc ions is affected by membrane environment and magnesium ions.

Rat matrix-induced alkaline phosphatase is an enzyme which requires magnesium and zinc ions for its maximal activity. Two Zn(II) ions and one Mg(II) ion are bound to each subunit of native dimeric enzyme. The presence of magnesium ion (10-100 microM) or zinc ion (7-20 nM) alone is sufficient to stimulate apoenzyme activity. However maximal activity (264 U/mg) requires the presence of both ions. Binding of Zn(II) ions to the Mg(II) binding site causes a strong inhibition of the apoenzyme while the binding of Mg(II) on Zn(II) binding site is not sufficient to stimulate PNPPase activity of the apoenzyme. Binding of both ions to the enzyme molecule did not change the apparent dissociation constant for PNPP hydrolysis.

1. Solubilized and membrane-bound alkaline phosphatase showed Michaelis-Menten behavior in a wide range of different substrate concentrations. 2. Membrane-bound alkaline phosphatase has a molecular weight of 130,000 and its minimum active configuration comprises two identical subunits of about 65,000. 3. The two forms of the enzyme behave similarly with respect to NaCl, urea and guanidine HCl. 4. Catalytic groups have pK values of about 8.5 and 9.7 for both membrane-bound and solubilized enzyme.