Purification and properties of human alkaline phosphatase from meconium.

Abstract

Human alkaline phosphatase was purified from meconium by treatment with cetylpyridinium chloride, followed by diethylaminoethyl (DEAE)-cellulose and CM-cellulose chromatography, Sephadex G-200 gel filtration and DEAE-Sephadex A-50 chromatography. The homogeneity of the purified enzyme was demonstrated by disc electrophoresis and immunological investigation. The molecular weight of the purified meconial alkaline phosphatase was 155000 and the enzyme was composed of two subunits of equal molecular weight. The optimum pH was found to be 10.0 and the enzyme was stable over the pH range of 4-10. The Km value was 2.2mM for p-nitrophenylphosphate as a substrate. The isoelectric point was pH 4.0, and the purified enzyme was inhibited by N-bromosuccinimide (NBS), o-phenanthroline, ethylenediaminetetraacetic acid (EDTA) and L-phenylalanine. Meconial alkaline phosphatase obtained by our method contained 2 g-atoms of zinc/mole of enzyme. The enzymic properties of the purified meconial alkaline phosphatase were compared with those of adult intestinal alkaline phosphatase.