Characterization of a calcium-dependent protein kinase from Arachis hypogea (groundnut) seeds

Abstract

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 μM). 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 μM). 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 μM) and calmidazolium (IC₅₀ = 10 μM), 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.