A second member of the novel Ca(2+)-dependent protein kinase family from Paramecium tetraurelia. Purification and characterization.
The ciliated protozoan Paramecium tetraurelia contains two protein kinase activities (CaPK-1 and CaPK-2) that are dependent on Ca2+ (Gundersen, R. E., and Nelson, D. L. (1987) J. Biol. Chem. 262, 4602-4609). We purified Ca(2+)-dependent protein kinase-1 (CaPK-1) 1,000-fold from the EGTA-extracted soluble fractions of Paramecium. The purified enzyme was a single polypeptide of 52 kDa on SDS-polyacrylamide gel electrophoresis with a native molecular mass of 60,000, suggesting that the active enzyme is a monomer. The purified kinase used casein as the best substrate in vitro, and its activity was absolutely dependent on Ca2+. The physical, catalytic and regulatory properties were clearly distinct from those of casein kinase, protein kinase C, and Ca2+/calmodulin-dependent protein kinases. CaPK-1 was half maximally activated by submicromolar (0.2 microM) free Ca2+, and the purified kinase bound Ca2+ in a blot overlay assay. CaPK-1 and the previously characterized CaPK-2 were biochemically and immunologically different enzymes sharing a similar activation mechanism. CaPK-1 and CaPK-2 appear to be members of a new family of Ca(2+)-dependent protein kinases. A protein immunologically related to the CaPKs was also detected in rat brain.
Son
M
M
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Gundersen
R E
RE
Nelson
D L
DL
D000818:Animals; D002384:Catalysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D016805:Paramecium tetraurelia / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases