Affinity purification and some molecular properties of human liver alkaline phosphatase

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sod...

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Bibliographic Details
Published in:Biochemical journal 1976-06, Vol.155 (3), p.653-660
Main Authors: Trépanier, J M, Seargeant, L E, Stinson, R A
Format: Article
Language:English
Subjects:
Alkaline Phosphatase - isolation & purification
Chromatography, Affinity
Chromatography, DEAE-Cellulose
Chromatography, Gel
Electrophoresis, Polyacrylamide Gel
Humans
Isoelectric Focusing
Liver - enzymology
Molecular Weight
Spectrophotometry
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Summary:Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj1550653