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...

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Bibliographic Details
Published in:Journal of dairy science 1992-12, Vol.75 (12), p.3394-3401
Main Authors: Bingham, E.W. (ARS, USDA, Eastern Regional Research Center, Philadelphia, PA), Garver, K, Powlen, D
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
Alkaline Phosphatase - antagonists & inhibitors
Alkaline Phosphatase - isolation & purification
Alkaline Phosphatase - metabolism
Animals
CATION
CATIONES
Cations, Divalent
Cattle - metabolism
Chromatography
CITOPLASMA
Cytoplasm - enzymology
CYTOPLASME
Electrophoresis, Polyacrylamide Gel
Female
FOSFATASA ALCALINA
GLANDE MAMMAIRE
GLANDULAS MAMARIAS
Hydrogen-Ion Concentration
INHIBIDORES DE ENZIMAS
INHIBITEUR D'ENZYME
LACTACION
LACTATION
Lactation - physiology
Mammary Glands, Animal - enzymology
Mammary Glands, Animal - ultrastructure
MEDIO DE CULTIVO
METAL
METALES
Metals - pharmacology
Microsomes - enzymology
MILIEU DE CULTURE
ORGANITE CELLULAIRE
ORGANULOS CITOPLASMATICOS
PHOSPHATASE ALCALINE
PURIFICACION
PURIFICATION
Substrate Specificity
VACAS LECHERAS
VACHE LAITIERE
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Summary: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)
ISSN:0022-0302
1525-3198
DOI:10.3168/jds.S0022-0302(92)78115-6