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-lOO 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 OOO± lOOOO 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 Zn 2 + ions are necessary for hydrolytic activity.Treatment with neuraminidase 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°C for 15 min. The activation energy, determined from a linear Arrhenius plot, was 50·1 kJmol-'.

Intraluminal injections of lectins, including concanavalin A (Con A), wheatgerm lectin, and soybean lectin, Con A-Sepharose 4B beads, calcium ionophore A23187 or peanut oil into the left uterine horns of mice on day 4 of pseudopregnancy induced the formation of deciduomata and significantly increased the weight and alkaline phosphatase activity of uterine tissue on day 7 of pseudopregnancy. In contrast, injections of these materials into the uterine horns of non-pseudopregnant mice that had not been previously mated failed to induce similar responses.

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Most of the alkaline phosphatase activity in the mouse uterus during early pregnancy was found to be membrane-bound and was associated with particulate material when homogenates were centrifuged at 105000 g. The activity of the enzyme increased in both the particulate and cytosol fractions of uterine homogenates during early pregnancy to reach maximum values on day 7 of pregnancy.

Uterine horns from unmated, pseudopregnant (days 1-5) and pregnant (days 4 and 5) mice were dissected and treated with collagenase (5 mg/ml) in a Ringer solution to isolate viable endometrial cells with maximal recovery of the luminal epithelium.