Purification and characterization of a Ca(2+)-dependent protein kinase from sandalwood (Santalum album L.): evidence for Ca(2+)-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 Ca(2+), K(0.5) (apparent binding constant) for Ca(2+)-activation of substrate phosphorylation activity being 0.7 microM and for autophosphorylation activity approximately 50 nM. Ca(2+)-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC(50) for W7=6 microM, for W5=46 microM) 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.
Anil
V S
VS
Department of Biochemistry, Indian Institute of Science, Bangalore-560 012, India.
Sankara Rao
K
K
D000596:Amino Acids / Q000032:analysis; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009994:Osmolar Concentration; D010766:Phosphorylation; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D000596:Amino Acids; D002118:Calcium; D002147:Calmodulin; D004791:Enzyme Inhibitors; D008274:Magnesium; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases