Purification and Characterization of Alkaline Phosphatase from Plasma Membranes of Rat Ascites Hepatoma1

Abstract

Alkaline phosphatase was purified from plasma membranes of rat ascites hepatoma AH-130, the homogenate of which had 50-fold higher specific activity than that found in the liver homogenate. The presence of Triton X-100, 0.5%, was essential to avoid its aggregation and to stabilize its activity. The purified enzyme, a glycoprotein, was homogeneous in poly-acrylamide gel electrophoresis. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated a protein molecular weight of 140,000. The addition of β-mercaptoethanol caused the dissociation of the alkaline phosphatase into two subunits of identical molecular weight, 72,000. Isoelectric focusing revealed that the pI of this enzyme is 4.7. The pH optimum for the purified enzyme was 10.5 or higher with p-nitrophenylphosphate, and slightly lower pH values (pH 9.5–10.2) were obtained when other substrates were used. Of the substrates tested, p-nitrophenylphosphate (K^m=0.3 mm) was most rapidly hydrolyzed. V_max values of other substrates relative to that of p-nitrophenyIphosphate were as follows; β-glycerophosphate, 76% 5′-TMP, 82%; 5′-AMP, 62%; 5′-IMP, 43%; glucose-6-phosphate, 39% ADP, 36%; and ATP, 15%. More than 90% of the activity of the purified enzyme was irreversibly lost when it was heated at 55°C for 30 min, or exposed either to 10 mM β-mercaptoethanol for 10 min, to 3 m urea for 30 min, or to an acidic pH below pH 5.0 for 2 h. Of the effects by divalent cations, Mg²⁺ activated the enzyme by 20% whereas Zn²⁺ strongly inhibited it by 95% at 0.5 mM. EDTA at higher than 1 mM inactivated the enzyme irreversibly, although the effect of EDTA at lower than 0.1 mM was reversible by the addition of divalent cations, particularly by Mg²⁺. The enzyme was most strongly inhibited by L-histidine among the amino acids tested, and also strongly inhibited by imidazole. These results suggest that alkaline phosphatase of rat hepatoma AH-130 is very similar to that of rat liver in most of the properties reported so far.