Purification and properties of alkaline phosphatase in the lactating bovine mammary gland.
Alkaline phosphatase has been purified 1400-fold from homogenates of lactating bovine mammary tissue. The purification procedure included subcellular fractionation, solubilization with butanol, fractionation with acetone, chromatography on concanavalin A-Sepharose, DEAE cellulose, DEAE-Sephadex, and gel filtration on Sephadex G-200. The enzyme activity was measured with the substrate p-nitrophenylphosphate in three buffers, and the maximum rate occurred at pH 10. For maximum activity, Mg2+ was required. Substrate specificity studies at three pH values indicated that the enzyme had broad specificity. It catalyzed the hydrolysis of aliphatic and aromatic phosphates and pyrophosphates, but the phosphoprotein beta-casein was a poor substrate. Potent inhibitors of the enzyme were levamisole and sulfhydryl reagents (2-mercaptoethanol, dithiothreitol, and cysteine).
Bingham
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Agricultural Research Service, USDA Eastern Regional Research Center, Philadelphia, PA 19118.
Garver
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Powlen
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D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent; D002417:Cattle / Q000378:metabolism*; D002845:Chromatography; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006863:Hydrogen-Ion Concentration; D007774:Lactation / Q000502:physiology*; D008321:Mammary Glands, Animal / Q000201:enzymology* / Q000648:ultrastructure; D008670:Metals / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D013379:Substrate Specificity
D002413:Cations, Divalent; D008670:Metals; D000469:Alkaline Phosphatase