Ca(2+)-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: partial purification and Ca(2+)-dependent association of the enzyme to the microsomes.
Ca(2+)-dependent protein kinase (CDPK) was purified 900-fold from the soluble fraction of Dunaliella tertiolecta cells by ammonium sulfate precipitation, DEAE-Toyopearl, phenyl-Sepharose, and hydroxylapatite column chromatography. The CDPK was activated by micromolar concentration of Ca2+ and required neither calmodulin nor phospholipids for its activation. The enzyme phosphorylated casein, myosin light chain, and histone type III-S (histone H-1), but did not phosphorylate protamine and phosvitin. The Km values for ATP and casein were 11 microM and 300 micrograms/ml, respectively. Phosphorylation of casein was inhibited by calmodulin antagonists, calmidazolium, trifluoperazine, and compound 48/80, but not affected by calmodulin. CDPK bound to phenyl-Sepharose in the presence of Ca2+ and was eluted by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca2+ and released in the presence of EGTA, suggesting the possibility of in vivo Ca(2+)-dependent association of the enzyme. The enzyme phosphorylated many proteins in the microsomes but few in the cytosol, if at all.
Yuasa
T
T
Institute of Applied Microbiology, University of Tokyo, Japan.
Muto
S
S
D002147:Calmodulin / Q000494:pharmacology; D000460:Chlorophyta / Q000201:enzymology*; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004789:Enzyme Activation; D006882:Hydroxyapatites; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D010743:Phospholipids / Q000494:pharmacology; D011073:Polyamines / Q000494:pharmacology; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D002147:Calmodulin; D017886:Durapatite; D006882:Hydroxyapatites; D010743:Phospholipids; D011073:Polyamines; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases