Expression and characterization of a recombinant maize CK-2 alpha subunit.
CKIIB, one of the CK-2 like enzymes which have been isolated from maize, has been shown to be a monomeric enzyme that cross-reacts with anti CK-2 alpha specific antibodies suggesting a possible relationship between the two proteins (Dobrowolska et al. (1992) Eur. J. Biochem. 204, 299-303). In order to support the immunological data also by biochemical and biophysical experiments the availability of a recombinant CK-2 alpha from maize was a prerequisite. A maize cDNA clone of maize CK-2 alpha was expressed in the bacterial strain BL21 (DE3). The recombinant protein was purified to homogeneity; its molecular mass on one-dimensional SDS PAGE was estimated to be 36.5 kDa. The calculated molecular mass according to the amino acid composition is 39,228 Da (332 amino acids). The recombinant maize CK-2 alpha (rmCK-2 alpha) exhibited mostly the same properties as the recombinant human CK-2 alpha (rhCK-2 alpha). In several respects it behaved differently from CKIIB, thus supporting the notion that either CKIIB is encoded by another gene or it undergoes extensive posttranscriptional and/or posttranslational alterations. Three observations in particular disprove any close relatedness between CKIIB and rmCK-2 alpha, namely: (a) the phosphorylation of calmodulin by CKIIB is dramatically stimulated by polylysine, whereas polylysine inhibits rather than stimulating the phosphorylation of calmodulin by rmCK-2 alpha (and by rhCK-2 alpha). (b) Addition of rhCK-2 beta has no significant influence on the stimulation of the calmodulin phosphorylation by CKIIB whereas in the case of rmCK-2 alpha and rhCK-2 alpha addition of rhCK-2 beta is required for optimal stimulation by polylysine. (c) CKIIB does not self-assemble with rhCK-2 beta to form a high molecular mass complex as it is demonstrated for rmCK-2 alpha.
Boldyreff
B
B
Institut für Humangenetik, Universität des Saarlandes, Homburg, Germany.
Meggio
F
F
Dobrowolska
G
G
Pinna
L A
LA
Issinger
O G
OG
D000595:Amino Acid Sequence; D001483:Base Sequence; D002147:Calmodulin; D047388:Casein Kinases; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011107:Polylysine; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D002147:Calmodulin; D011994:Recombinant Proteins; D011107:Polylysine; D011494:Protein Kinases; D047388:Casein Kinases