Activity of the E 75 E 76 mutant of the α subunit of casein kinase II from Xenopus laevis

• Published in: FEBS letters 1994-02, Vol.339 (1-2), p.93-96
• Main Authors: Gatica, Marta, Jedlicki, Ana, Allende, Catherine C., Allende, Jorge E.
• Format: Article
• Language: English
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(94)80392-7

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 Lys 75 and Lys 76 , 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. K m 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 αE 75 E 76 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.