Purification and properties of alkaline phosphatase from rat chloroma.

Activity was determined by the rate of hydrolysis of 13-glycerophosphate on p-nitrophenyl phosphate (8). The determination with sodium 13-glycerophosphate was carried out as follows. The reaction mixture consisted of 0.05 M Tnis-HC1 (pH 10), 0.023 M sodium 13-glycerophosphate, 0.004 M MgCl2, and enzyme in a final volume of 2 ml. After incubation at 37° for 30 mm, 2 ml of 10% tnicbloroacetic acid were added. Inorganic phosphate was determined in the supernatant by the method of Fiske and SubbaRow (7). Activity was expressed as pmoles P1/30 min/ml of enzyme solution or per mg of protein. Protein concentration was determined by the method of Lowry ci' al. (10), with crystalline bovine serum albumin as standard. The rate of hydrolysis of p-nitrophenyl phosphate was determined as described by Harkness (9). The activity was expressed as units/ml, 1 unit being defined as a change in absorbance at 410 mp of 1.0/mm. Specific activity was expressed as units/mg of protein. Disc electrophoresis on polyacrylamide gel (1 5) was performed in a Canalco apparatus as previously described (28). At the termination of the electrophoretic run, the gels were stained either for protein or for AP activity. Protein was stained with 1% Amido black, and excess stain was removed by washing in 7.5% acetic acid. Enzymatic activity was located as described by Burstone (3) with a-naphthyl phosphate, 0.2 mg/mi, and fast red violet B, 1 mg/mI, in 0.05 M Tnis-HC1 buffer, pH 9.0.