Association of Placental Alkaline Phosphatase Activity with Preparations of the Human Placental Inhibitor to Hemagglutination by H-1 Virus

Usategui-Gomez, M.; Ghosh, Nimai K.; Toolan, Helene Wallace

doi:10.3181/00379727-130-33735

Cited by 6 publications

(1 citation statement)

References 6 publications

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“…However, as has been found for other sialic acid-containing phosphatases Bachhawat 1968, 1970;Mulivor et al 1978;Otani et al 1978), desialylation failed to alter the activity or the catalytic properties of the uterine enzyme suggesting that the sialic acid residues do not constitute a part of the active centre of the enzyme. Attempts to elucidate the functional significance of sialic acid in alkaline phosphatase enzymes and other glycoproteins have shown that this component may nevertheless influence antigenic specificity (Badger and Sussman 1976) and the ability of the protein to inhibit the agglutination of viruses (Usategui-Gomez et al 1969).…”

Section: Arrhenius Plot and Activation Energymentioning

confidence: 99%

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

Murdoch¹,

Buxton²,

Kay³

1980

Aust. Jnl. Of Bio. Sci.

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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-'.

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