PLANT PHYSIOLOGY , Vol 104, Issue 3 961-969, Copyright © 1994 by American Society of Plant Biologists
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CELL BIOLOGY AND SIGNAL TRANSDUCTION |
Characterization of a Calcium-Dependent Protein Kinase from Arachis hypogea (Groundnut) Seeds
M. DasGupta
Department of Biophysics, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India
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 [mu]M). 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 [mu]M). 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 [mu]M) and calmidazolium (IC50 = 10 [mu]M),
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.
