title: Biochemical characterization of a calcium-sensitive protein kinase LeCPK2 from tomato.
abstract: LeCPK2 (GenBank GQ205414), a versatile calcium-dependent protein kinase (CDPK or CPK) gene was isolated from tomato in our previous study. In this study, the biochemical properties of LeCPK2 were further investigated. To examine the role of the C-terminal calmodulin-like domain (CLD) of LeCPK2 with respect to Ca2+ activation, the kinase activities of recombinant full-length and truncated LeCPK2 were measured by Kinase-Glo Luminescent kinase assay (Promega). The results showed that LeCPK2 activity was Ca(2+)-dependent and the C-terminal CLD of 161 residues was essential for the activation of LeCPK2. The activity of LeCPK2 was sharply stimulated by Ca2+ with K0.5 (concentration of Ca2+ for half-maximal activity) of 48.8 and 45.5 nM with substrate histone IIIs and syntide 2, respectively. The optimal concentration of Mg2+ for LeCPK2 activity was 20 and 10 mM for substrate histone IIIs and syntide 2, respectively. The K(m) value of LeCPK2 towards histone IIIs and syntide 2 was 44.9 microg/ml and 89.52 microM, respectively. The determination of biochemical properties of LeCPK2 would provide some clues on how its activity was regulated in vivo.
authors.affiliation: Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Chang
authors.forename: Gui
authors.initials: G
authors.lastname: Fu
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
authors.forename: Jiahong
authors.initials: J
authors.lastname: Zhu
authors.forename: Zhili
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D002122:Calcium Chloride / Q000737:chemistry*; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D015636:Magnesium Chloride / Q000737:chemistry*; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D002122:Calcium Chloride; D015636:Magnesium Chloride; D011494:Protein Kinases; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases