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 mu mol mg(-1) min(-1), K-m for ATP: 67 mu M, K-m for syntide 2: 15 mu M). The kinase showed an optimum pH of 6.8 and a marked dependence on low-micromolar Ca2+ concentrations (K-d: 0.77 mu M). 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 Ca2+-dependent manner.