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Purification and Properties of Adenylate Pyrophosphorylase from Ehrlich Ascites Tumor Cells

From the ¹ From University of Alberta Cancer Research Unit (McEachern Laboratory) and the Department of Biochemistry, Edmonton, Alberta, Canada

Adenylate pyrophosphorylase was purified 65-fold from Ehrlich ascites carcinoma cells by a procedure involving acid treatment, carboxymethyl Sephadex, diethylaminoethyl Sephadex, and precipitation with (NH₄)₂SO₄. The specific activity of the most highly purified preparation was more than twice that previously reported. Further purification was attempted but was unsuccessful because of the instability of this enzyme.

The partially purified enzyme was stable when lyophilized, but unstable in solution unless sulfate ion was present. Sulfate ion also partially protected the enzyme from heat denaturation. Adenylate pyrophosphorylase was stable between pH 5 and pH 11 but was completely inactivated below pH 4. It was partially inhibited by sodium ion, sulfate, phosphate, and several carboxylic acids.

An absolute requirement for a divalent cation was demonstrated, and Ca²⁺, Mn²⁺, and Mg²⁺ all supported high initial rates of reaction. Co²⁺, Ni²⁺, and Zn²⁺ gave lower but still substantial rates.

The isoelectric point of AMP pyrophosphorylase was found to be pH 5.1.

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