Modulation of activity of human alkaline phosphatases by Mg 2+ and thiol compounds

Abstract

Interaction of purified human liver and placental alkaline phosphatases (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) with sulfhydryl groups, sulfhydryl reagents, and Mg 2+ were studied. l-Cysteine (0.1 mmol/l) or Mg 2+ activated the liver enzyme 4–5-fold and the placental enzyme 2–3-fold, with optimal pH 7.5–8.0; these activations were not additive. l-Cysteine (2 mmol/l) inhibited both enzymes maximally at pH > 9.0 ; phosphate protected the enzymes. S- Methylcysteine had little effect, with or without Mg 2+. Inhibition by sulfur-containing compounds paralleled their ability to bind Zn 2+. Fluoresceine mercury acetate (specific for sulfhydryl groups) inhibited the isoenzymes, whereas iodoacetic acid, iodoacetamide, dithionitrobenzoic acid, and p- chloromercuribenzoate had little effect. The inhibition was reversed by l-cysteine and only slightly protected by inorganic phosphate. Thus, there are two sites on human liver and placental alkaline phosphatase that interact with l-cysteine; a Mg 2+-binding site, which results in activation, and a site that involves one or both of the bound Zn 2+ ions and results in inactivation. Both enzymes have a protected essential thiol group.