Purification and characterization of a Ca 2+-dependent protein kinase from sandalwood ( Santalum album L.): evidence for Ca 2+-induced conformational changes

Abstract

An early development-specific soluble 55 kDa Ca 2+-dependent protein kinase has been purified to homogeneity from sandalwood somatic embryos and biochemically characterized. The purified enzyme, swCDPK, resolved into a single band on 10% polyacrylamide gels, both under denaturing and non-denaturing conditions. swCDPK activity was strictly dependent on Ca 2+, K 0.5 (apparent binding constant) for Ca 2+-activation of substrate phosphorylation activity being 0.7 μM and for autophosphorylation activity ∼50 nM. Ca 2+-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC 50 for W7=6 μM, for W5=46 μM) and cross-reaction with polyclonal antibodies directed against the CaM-like domain of soybean CDPK, confirmed the presence of an endogenous CaM-like domain in the purified enzyme. Kinetic studies revealed a K m value of 1.3 mg/ml for histone III-S and a V max value of 0.1 nmol min −1 mg −1. The enzyme exhibited high specificity for ATP with a K m value of 10 nM. Titration with calcium resulted in the enhancement of intrinsic emission fluorescence of swCDPK and a shift in the λ max emission from tryptophan residues. A reduction in the efficiency of non-radiative energy transfer from tyrosine to tryptophan residues was also observed. These are taken as evidence for the occurrence of Ca 2+-induced conformational change in swCDPK. The emission spectral properties of swCDPK in conjunction with Ca 2+ levels required for autophosphorylation and substrate phosphorylation help understand mode of Ca 2+ activation of this enzyme.