Activity of the E75E76 mutant of the α subunit of casein kinase II from Xenopus laevis

Abstract

The cDNA gene coding for the α 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 α 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 β subunit. Inhibition by quercetin and by 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole was also the same for mutant and wild type subunits. However, the CKII αE75E76 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. © 1994.