Boldyreff, B5; James, P7; Staudenmann, W5; Issinger, O G8
1 Faculty of Science, SDU2 Det Sundhedsvidenskabelige Fakultet, SDU3 Geriatrics, Department of Clinical Research, Det Sundhedsvidenskabelige Fakultet, SDU4 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU5 unknown6 Dept. Clinical Biochemistry and Pharmacology, Department of Public Health, Det Sundhedsvidenskabelige Fakultet, SDU7 Dept. Clinical Biochemistry and Pharmacology, Department of Public Health, Det Sundhedsvidenskabelige Fakultet, SDU8 Department of Biochemistry and Molecular Biology, Faculty of Science, SDU
Human casein kinase-2 (CK-2) subunits alpha and beta were bicistronically expressed in bacteria. The recombinant holoenzyme shared all investigated properties with the native CK-2 from mammalian sources (rat liver, Krebs II mouse ascites tumour cells). Contrary to recombinant human CK-2 produced by self-assembly in vitro, the bicistronically expressed beta subunit was autophosphorylated during formation of the holoenzyme in bacteria. Electrospray ionisation mass spectrometry (ESI) revealed Ser2 (second amino acid, first serine) as the only phosphate acceptor site. Kinetic data obtained with either the phosphorylated or the unphosphorylated form of CK-2 did not differ significantly, suggesting that the autophosphorylation had no influence on the kinetic parameters Km and Vmax. In parallel, native rat liver CK-2 beta subunit was shown to incorporate 0.1 mol phosphate/mol holoenzyme, which suggests that the enzyme is already heavily phosphorylated. ESI analysis also revealed Ser2 as the only phosphorylated residue at the amino terminus. In the case of both recombinant human CK-2 and native rat liver CK-2, treatment with alkaline phosphatase readily reversed the phosphorylated form of the beta subunit to the faster migrating dephosphorylated polypeptide.
European Journal of Biochemistry, 1993, Vol 218, Issue 2, p. 515-521