Characterization of a calcium-dependent protein kinase from Arachis hypogea (groundnut) seeds.
A calcium-dependent protein serine/threonine kinase (GnCDPK) has been detected in groundnut (Arachis hypogea) seeds that specifically phosphorylates a peptide (MLCpep) representing the phosphate-accepting domain of smooth muscle myosin light chains. GnCDPK has been purified to near homogeneity from the soluble fraction of groundnut seeds by ammonium sulfate precipitation, Q Sepharose, Blue Sepharose, and Sephacryl 300 chromatography. The molecular weight of GnCDPK is estimated to be 53,000. Enzyme activity is stimulated about 100-fold in the presence of free Ca2+ (concentration required for half-maximal activation = 0.5 microM). GnCDPK is capable of binding 45Ca2+ ions directly in an electroblot, indicating it to be a calcium-binding protein. Phosphorylation of MLCpep is found to be optimal at an alkaline pH range (pH 9-10). Unlike all other calcium-dependent protein kinases reported from higher plants, GnCDPK does not accept casein or histones as substrate. Sequences related to MLCpep (> 60% homologous) that are present in myosin light chains from skeletal muscles of chicken and rabbit also fail to act as a substrate for GnCDPK. In contrast to the Ca2+/calmodulin dependence of myosin light chain kinases, GnCDPK activity is not affected by the presence of exogenous calmodulin (1-10 microM). However, enzyme activity is considerably inhibited in the presence of calmodulin antagonists like N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (concentration required for 50% inhibition [IC50] = 30 microM) and calmidazolium (IC50 = 10 microM), indicating an endogenous calmodulin structure to be present in GnCDPK. The probability of GnCDPK being a bona fide plant myosin light chain kinase is discussed.
DasGupta
M
M
Department of Biophysics, University of Delhi South Campus, India.
D000595:Amino Acid Sequence; D000818:Animals; D010367:Arachis / Q000201:enzymology* / Q000235:genetics; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D002118:Calcium; D002147:Calmodulin; D008274:Magnesium; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases