Partial purification and characterization of a Ca(2+)-dependent protein kinase from the green alga, Dunaliella salina.
A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina.  The enzyme was activated at free Ca2+ concentrations above 10(-7) molar. and half-maximal activation was at about 3 x 10(-7) molar.  The optimum pH for its Ca(2+)-dependent activity was 7.5.  The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca2+.  The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar.  Immunoassay showed some calmodulin was present in the kinase preparations.  However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca2+ in gel assays after being electrophoretically separated from calmodulin by two different methods.  This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure.  Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1.  By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.
Guo Y-L
Department of Botany, The University of Texas at Austin 78713.
Roux
S J
SJ
D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry; D000460:Chlorophyta / Q000201:enzymology*; D010743:Phospholipids / Q000737:chemistry; D010766:Phosphorylation; D010940:Plant Proteins; D011494:Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D002147:Calmodulin; D010743:Phospholipids; D010940:Plant Proteins; D011494:Protein Kinases; D002118:Calcium