KINETIC-STUDIES ON THE MEMBRANE FORM OF INTESTINAL ALKALINE-PHOSPHATASE

We have purified different membrane and soluble forms of alkaline phos phatase from human placenta and bovine intestine. The enzymes will be used as markers in immunoconjugates and/or as model for membrane enzym e studies. The membrane form of alkaline phosphatase extracted from bo vine intestine was purified on Q-Sepharose and on L-histidyldiazobenzy l-phosphonic acid-agarose columns to remove phosphodiesterase activity . The purified enzyme had a molecular mass of 61 kDa, Km of 1208 mu M, and Vmax 240 mu mol pNP/min when assayed in 1 M diethanolamine, 0.5 m M MgCl2 buffer, pH 9.8, containing 10 to 2250 mu M of pNPP at 37 degre es C. In the present investigation we studied the effect of salts and inositol derivatives on this enzyme activity, which was found to depen d on 0.5 mM Mg2+, and to be fully inhibited by 1.2 mM Hg2+. Vanadate ( 0.5 mM) and Zn2+ (0.5 mM) reduced the Km value by 43% and 84%, respect ively. Inositol (2 mM) and inositoi-2-monophosphate (2 mM) reduced the activity by 23% and 17%. Inositol-1-monophosphate (0.5 mM) and cyclic -inositol-(1:2)-monophosphate (0.5 mM) enhanced their Km value by at l east 30% compared to p-nitrophenylphosphate.