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

Anil, Veena S and Rao, Sankara K (2001) Purification and characterization of a $Ca^{2+}$-dependent protein kinase from sandalwood (Santalum album L.): evidence for $Ca^{2+}$-induced conformational changes. In: Phytochemistry, 58 (2). pp. 203-212.

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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 $0.7 \mu M$ and for autophosphorylation activity $\sim 50 \hspace{2mm} nM$. $Ca^{2+}$-dependence for activation, CaM-independence, inhibition by CaM-antagonist $(IC_{50}$ for W7=6 $\mu M$, for W5=46 $\mu M)$ 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 $K_m$ 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 $Ca^{2+}$-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.

Item Type:	Journal Article
Publication:	Phytochemistry
Publisher:	Elsevier
Additional Information:	Copyright of this article belongs to Elsevier.
Keywords:	Santalum album L.;Santalaceae;Sandalwood;Signal transduction;Protein purification;Calmodulin-like domain protein kinase;Ca2+-dependent protein kinase;Enzyme activation;Conformational changes
Department/Centre:	Division of Biological Sciences > Biochemistry
Date Deposited:	28 Sep 2007
Last Modified:	19 Sep 2010 04:39
URI:	http://eprints.iisc.ac.in/id/eprint/11974

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