Overexpression, purification and characterization of Dictyostelium calcineurin A.
The catalytic subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin A) was overexpressed about 50-fold in Dictyostelium discoideum cells transformed with a vector containing the cDNA for D. discoideum calcineurin A under control of the actin-6 promoter. In crude lysates from the overexpressing cell line, high Ca2+/calmodulin-stimulated phosphatase activity was detected. Calcineurin A was purified by anion exchange chromatography and calmodulin-Sepharose affinity chromatography, and the enzymatic activity of the isolated protein was characterized. Its phosphatase activity was strictly dependent on the addition of divalent metal ions such as Mg2+ or Mn2+. Disulphide-reducing agents increased the activity more than 10-fold. Ca2+/calmodulin stimulated the activity by a factor of 2.5-5. Despite the high extra Ca2+/calmodulin-dependent phosphatase activity, the overexpressing cell line showed no phenotypic aberrations.
Fakultät fur Biologie, Universität Konstanz, Germany.
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Hellstern
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Dammann
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Husain
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Mutzel
D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D019703:Calcineurin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002147:Calmodulin / Q000494:pharmacology; D002364:Caseins / Q000378:metabolism; D002413:Cations, Divalent; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D018076:DNA, Complementary; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014170:Transformation, Genetic
D000814:Aniline Compounds; D065095:Calcineurin Inhibitors; D002147:Calmodulin; D002364:Caseins; D002413:Cations, Divalent; D002712:Chlorides; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D017895:Manganese Compounds; D009943:Organophosphorus Compounds; D010748:Phosphopeptides; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents; D015636:Magnesium Chloride; D019319:Okadaic Acid; C068798:4-aminophenylphosphate; D019703:Calcineurin; C025340:manganese chloride; D004229:Dithiothreitol