ACTIVITY OF THE E(75)E(76) MUTANT OF THE A SUBUNIT OF CASEIN KINASE-II FROM XENOPUS-LAEVIS

The cDNA gene coding for the a subunit of Xenopus laevis casein kinase II was mutated using the overlap extension PCR method. The mutation s ubstituted glutamic acids for Lys(75) and Lys(76), changing the charge distribution of a very basic sequence found in the alpha-subunit. Exp ression of the mutated cDNA in a pT7-7 vector in E. coli yielded an ac tive mutant recombinant protein that was extensively purified. This mu tant was not significantly affected in its app. K-m for casein or a mo del peptide substrate, nor in its interaction with the activating P su bunit. Inhibition by quercetin and by 5,6-dichloro-1-beta-D-ribofurano syl benzimidazole was also the same for mutant and wild type subunits. However, the CKII alpha E(75)E(76) mutant was at least one order of m agnitude less sensitive to inhibition by polyanionic inhibitors such a s heparin, poly U, copolyglutamic acid:tyrosine (4:1) and 2,3 diphosph oglycerate.