Activity of the E75E76 mutant of the alpha subunit of casein kinase II from Xenopus laevis.
The cDNA gene coding for the alpha subunit of Xenopus laevis casein kinase II was mutated using the overlap extension PCR method. The mutation substituted glutamic acids for Lys75 and Lys76, changing the charge distribution of a very basic sequence found in the alpha subunit. Expression of the mutated cDNA in a pT7-7 vector in E. coli yielded an active mutant recombinant protein that was extensively purified. This mutant was not significantly affected in its app. Km for casein or a model peptide substrate, nor in its interaction with the activating beta subunit. Inhibition by quercetin and by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole was also the same for mutant and wild type subunits. However, the CKII alpha E75E76 mutant was at least one order of magnitude less sensitive to inhibition by polyanionic inhibitors such as heparin, poly U, copolyglutamic acid:tyrosine (4:1) and 2,3 diphosphoglycerate.
Gatica
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Departamento de Bioquímica, Facultad de Medicina, Universidad de Chile.
Jedlicki
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Allende
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Allende
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D047390:Casein Kinase II; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004563:Electrochemistry; D005971:Glutamates; D018698:Glutamic Acid; D008239:Lysine; D008969:Molecular Sequence Data; D009154:Mutation; D011072:Poly U / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011794:Quercetin / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014982:Xenopus laevis / Q000235:genetics
D018076:DNA, Complementary; D005971:Glutamates; D011994:Recombinant Proteins; D011072:Poly U; D018698:Glutamic Acid; D011794:Quercetin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D008239:Lysine