Partial purification and characterization of a Ca(2+)-dependent protein kinase from pea nuclei.
Almost all the Ca(2+)-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl.  This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca2+ and reaches half-maximal activation at about 3 x 10(-7) molar free Ca2+, without calmodulin.  It is a monomer with a molecular weight near 90,000.  It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly.  Its Ca(2+)-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine.  It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca(2+)-dependent protein kinases.  It is not stimulated by exogenous phospholipids or fatty acids.  In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.
Li
H
H
Department of Botany, The University of Texas at Austin 78713, USA.
Dauwalder
M
M
Roux
S J
SJ
D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology*; D002843:Chromatin / Q000737:chemistry; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism; D018532:Pisum sativum / Q000166:cytology / Q000201:enzymology*; D010766:Phosphorylation; D010940:Plant Proteins / Q000032:analysis / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D002118:Calcium; D002147:Calmodulin; D002843:Chromatin; D009687:Nuclear Proteins; D010940:Plant Proteins; D011493:Protein Kinase C; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases