Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes.
Several calcium-dependent protein kinases (CDPKs) are located in plant plasma membranes where they phosphorylate enzymes and transporters, like the H(+)-ATPase and water channels, thereby regulating their activities. In order to determine which kinases phosphorylate the H(+)-ATPase, a calcium-dependent kinase was purified from beetroot (Beta vulgaris L.) plasma membranes by anion-exchange chromatography, centrifugation in glycerol gradients and hydrophobic interaction chromatography. The kinetic parameters of this kinase were determined (V(max): 3.5 micromol mg(-1) min(-1), K(m) for ATP: 67 microM, K(m) for syntide 2: 15 microM). The kinase showed an optimum pH of 6.8 and a marked dependence on low-micromolar Ca(2+) concentrations (K(d): 0.77 microM). During the purification procedure, a 63-kDa protein with an isoelectric point of 4.7 was enriched. However, this protein was shown not to be a kinase by mass spectrometry. Kinase activity gels showed that a 50-kDa protein could be responsible for most of the activity in purified kinase preparations. This protein was confirmed to be a CDPK by mass spectrometry, possibly the red beet ortholog of rice CDPK2 and Arabidopsis thaliana CPK9, both found associated with membranes. This kinase was able to phosphorylate purified H(+)-ATPase in a Ca(2+)-dependent manner.
Lino
Bárbara
B
Departamento de Biotecnología y Bioquímica, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, Apartado postal 629, 36500 Irapuato Gto, México.
Carrillo-Rayas
M Teresa
MT
Chagolla
Alicia
A
González de la Vara
Luis E
LE
D027461:Beta vulgaris / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology*; D015180:Electrophoresis, Gel, Two-Dimensional; D007700:Kinetics; D010766:Phosphorylation; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D010940:Plant Proteins; D011494:Protein Kinases; D006180:Proton-Translocating ATPases; D017871:Calcium-Calmodulin-Dependent Protein Kinases