Purification and characterization of a Ca²⁺-dependent protein kinase from sandalwood (Santalum album L.): evidence for Ca²⁺-induced conformational changes

Abstract

An early development-specific soluble 55 kDa Ca²⁺-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 Ca²⁺, K_0.5 (apparent binding constant) for Ca²⁺-activation of substrate phosphorylation activity being 0.7 μ M and for autophosphorylation activity ~50 nM. Ca²⁺-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC⁵⁰ for W7=6 μ M, for W5=46 μ M) 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 Km 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 Ca²⁺-induced conformational change in swCDPK. The emission spectral properties of swCDPK in conjunction with Ca²⁺ levels required for autophosphorylation and substrate phosphorylation help understand mode of Ca²⁺ activation of this enzyme.