Purification and characterization of recombinant protein kinase CK2 from Zea mays expressed in Escherichia coli.
Recombinant protein kinase subunits rmCK2alpha-1 and rmCK2beta-1 from Zea mays were expressed separately in Escherichia coli and assembled to a fully active tetrameric holoenzyme complex in vitro. The obtained maize holoenzyme was purified to homogeneity, biochemically characterized, and compared to CK2 from human. Kinetic measurements of the recombinant maize holoenzyme (rmCK2) revealed k(cat) values for ATP and GTP of 4 and 2s(-1), respectively; whereas the recombinant maize catalytic subunit showed almost equal values for ATP and GTP, i.e., ca. 0.8s(-1). A comparison of the k(cat)/K(m) ratio between the maize holoenzyme and the catalytic subunit from CK2 maize shows that the incorporation of the catalytic subunit into the holoenzyme leads to a 14-fold activation in the case of ATP and 8-fold activation in the case of GTP. The maize holoenzyme is about 10 times more sensitive towards CK2 inhibitor heparin, on the other hand, it is stimulated only 0% by polylysine as compared to the human counterpart. The maize holoenzyme activity is more sensitive towards NaCl concentrations higher than those of rhCK2 and treatment with urea showed that rmCK2 holoenzyme was denatured more readily than the human holoenzyme.
Departamento de Genetica Molecular, IBMB, CID, C.S.I.C., Jordi Girona 18-26, 08034 Barcelona, Spain.
Marta
M
Riera
Montserrat
M
Pages
Olaf Georg
OG
Issinger
Barbara
B
Guerra
D000255:Adenosine Triphosphate / Q000378:metabolism; D015153:Blotting, Western; D047390:Casein Kinase II; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D011107:Polylysine / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry*; D012965:Sodium Chloride / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology; D003313:Zea mays / Q000201:enzymology*
D011994:Recombinant Proteins; D011107:Polylysine; D012965:Sodium Chloride; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D014508:Urea; D006493:Heparin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases