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

Abstract

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 Ca 2+ 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 K m values for ATP and casein were 11 μ m and 300 μg/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 Ca 2+ and was eluted by ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca 2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca 2+ 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.