Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.5 (5'-nucleotidase)
 3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A subcellular fractionation method to isolate simultaneously apical and basolateral plasma membrane fractions from the human adenocarcinoma cell line Caco-2, grown on filter supports, is described. The method employs sucrose-density-gradient centrifugation and differential precipitation. The apical membrane fraction was enriched 14-fold in sucrase-isomaltase and 21-fold in 5'-nucleotidase compared with the homogenate. The basolateral membrane fraction was enriched 20-fold relative to the homogenate in K(+)-stimulated p-nitrophenylphosphatase. Alkaline phosphatase was enriched 15-fold in the apical membrane fraction and 3-fold in the basolateral membrane fraction. Analytical density-gradient centrifugation showed that this enzyme was a true constituent of both fractions, and experiments measuring alkaline phosphatase release following treatment with phosphatidylinositol-specific phospholipase C showed that in both membrane fractions the enzyme was glycosyl-phosphatidylinositol-linked. There was very little contamination of either membrane fraction by marker enzymes of the Golgi complex, mitochondria or lysosomes. Both membrane fractions were greater than 10-fold purified with respect to the endoplasmic reticulum marker enzyme alpha-glucosidase. Protein composition analysis of purified plasma membrane fractions together with domain-specific cell surface biotinylation experiments revealed the presence of both common and unique integral membrane proteins in each plasma membrane domain. The post-synthetic transport of endogenous integral plasma membrane proteins was examined using the devised subcellular fractionation procedure in conjunction with pulse-chase labelling experiments and immunoprecipitation. Five common integral membrane proteins immunoprecipitated by an antiserum raised against a detergent extract of the apical plasma membrane fraction were delivered with the same time course to each cell-surface domain.
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PMID:The post-synthetic sorting of endogenous membrane proteins examined by the simultaneous purification of apical and basolateral plasma membrane fractions from Caco-2 cells. 131 18

Microvillus membrane vesicles from pig small intestine, isolated by hypotonic lysis, Mg2+ aggregation of contaminants and differential centrifugation, have been further purified by immunoadsorbent chromatography. The vesicles adhere to an immunoadsorbent prepared by coupling antibodies raised against three of the principal proteins of the brush border membrane (aminopeptidase, sucrase-isomaltase and lactase) to Sepharose 4B. After the contaminants are removed by washing, the adherent vesicles are released from the immunoabsorbent by applying shear forces. The purity of the immunoadsorbed vesicles has been established by electron microscopy and by measuring the activity of marker enzymes. The enrichment factor is 1.17 +/- 0.02 for aminopeptidase and 0.70 +/- 0.05 for 5'-nucleotidase. The contamination of the preparation before immunoadsorption constitutes 10% of the membrane protein and consists mainly of basolateral membrane fragments as judged from marker enzyme determinations and the lipid composition.
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PMID:Purification of microvillus membrane vesicles from pig small intestine by immunoadsorbent chromatography. 710 46