Mouse uterine alkaline phosphatase: improved purification by affinity chromatography and further characterization of the enzyme.
An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0.8% (v/v) Triton X-100 and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 000 +/- 10 000 was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme. Some metalloenzyme properties of the phosphatase were also studied and it was demonstrated that both Mg2+ and Zn2+ ions are necessary for hydrolytic activity. Treatment with neuraminidases retarded the anodal migration of the enzyme during electrophoresis on cellulose acetate membranes but did not influence its activity or catalytic properties. These results suggest that the uterine alkaline phosphatase is a sialoglycoprotein. The sialic acid residues, however, do not appear to constitute part of the active centre of the enzyme. In addition, the optimum pH for activity depended on substrate concentration and decreased with decreasing substrate concentration. Apparent Km values also depended on variations in pH and decreased with decreasing pH. Plots of pKm versus pH revealed a functional group with a pK value of 9.45. The enzyme also hydrolysed a variety of compounds having either phosphomonoester or pyrophosphate linkages and was inactivated after heating at 60 degrees C for 15 min. The activation energy, determined from a linear Arrhenius plot, was 50.1 kJ mol-1.
D000469:Alkaline Phosphatase / Q000032:analysis / Q000302:isolation & purification*; D000818:Animals; D001051:Apoenzymes / Q000302:isolation & purification; D002614:Chelating Agents / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D051379:Mice; D009439:Neuraminidase / Q000494:pharmacology; D011247:Pregnancy; D014599:Uterus / Q000201:enzymology*
D001051:Apoenzymes; D002614:Chelating Agents; D008670:Metals; D000469:Alkaline Phosphatase; D009439:Neuraminidase