Mouse Uterine Alkaline Phosphatase: Improved Purification by Affinity Chromatography and Further Characterization of the Enzyme

Abstract

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-'.

Genital Tract Proteins in the Male Rabbit: II. Alkaline Phosphatase - Enzyme Action and Site of Synthesis

“…-After being inhibited, different divalent metal ions were used for reactivation of the phosphatase, and most effective were Mgzt and ZnZt. This was also reported by Fernley -1971, Murdoch et al -1980, andRej andBretaudiere -1980. But no study dealt with the influence of CaZf; this metal ion caused the highest reactivation rate, rendering an enzyme activity even higher than the original. Reports on appearance of alkaline phosphatases in the seminal plasma already were given by Bern (1949) who studied cytochemically the phosphatase reactions of the testis, the epididymis and the vasa deferentia, and Bell and Lake (1962) who compared alkaline phosphatase activities of different species.…”

Behaviour and Properties of Membrane-Bound Mouse Uterine Alkaline Phosphatase During Early Pregnancy

“…Km (Michaelis constant) values, energy of activation values, and the effects of pH and temperature on the alkaline phosphatase activities of the various uterine preparations were determined as described by Murdoch et al (1980) using a 50 mM glycine-NaOH buffer system at pH 10•5.…”

“…Purified mouse uterine alkaline phosphatase was obtained as described in a previous paper (Murdoch et al 1980). When used in conjunction with the various sediment and supernatant fractions described above, purified preparations,were diluted with 0•25 M sucrose to ensure that the enzyme activity of all samples being studied si(hultaneously was approximately comparable.…”

A New Insulin Derivative for the Preparation of Insulin-Enzyme Conjugates: An Application in the Enzyme Immunoassay of Insulin

“…Human placental alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) was isolated from fresh placenta and purified by an affinity chromatography procedure (Murdoch et al 1980).…”