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

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 Ca2+, K-0.5 (apparent binding constant) for Ca2+-activation of substrate phosphorylation activity being 0.7 muM and for autophosphorylation activity similar to 50 nM. Ca2+-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC50 for W7=6 muM, for W5=46 muM) 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 lambda (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 Call levels required for autophosphorylation and substrate phosphorylation help understand mode of Ca2+ activation of this enzyme. (C) 2001 Elsevier Science Ltd. All rights reserved.