A comparative study of alkaline phosphatase in calcifying cartilage, odontoblasts and the enamel organ

Abstract

The enzyme alkaline phosphatase (AP) (EC 3.1.3.1) in three different calcification areas was studied by means of a spectrophotometric micro method using p-nitrophenylphosphate as a substrate. Rat maxillary incisor odontoblasts and enamel organ from the zones of matrix formation and maturation and tissue from rabbit metatarsal cartilage were allowed to react with the substrate in glycine-NaOH buffer at room temperature. The reaction was found to be linear for a minimum of 20 min. The pH optima for AP from these tissues were in the pH range of 10.0–10.3. In order to compare AP from the four calcification areas different parameters were studied. Heating at 56°C or 60°C for varying times revealed that the enzymes were almost completely inactivated after 10 min. Mg²⁺ ions activated the enzymes by about 25% at concentrations of 2.5 mM (enamel organ 1.25 mM); while only higher concentrations of Mg²⁺ had an inactivating effect, Ca²⁺ and PO ₄ ³⁻ ions were inactivating at varying concentrations. F⁻ ions show no effect on AP activity at concentrations below 250 mM (enamel organ 125 mM) but caused inactivation of the enzymes at about 50% at 1 M. EDTA was found to be a very effective AP inactivator at concentrations above 0.06 mM, whereas urea did not noticeably affect the enzyme reactions at concentrations below 1 M. At higher concentrations, inactivation was observed. In order to determine AP localization in the epiphyseal plate successive 40-μm-thick, freeze-sectioned slices were analyzed. The activity was highest nearest the zone of cartilage calcification and decreased towards the reserve cell zone. It was concluded that the same AP isoenzyme is present in these quite different calcification loci.