Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes |
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Authors: | Bárbara Lino M Teresa Carrillo-Rayas Alicia Chagolla Luis E González de la Vara |
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Institution: | (1) Departamento de Biotecnología y Bioquímica, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, Apartado postal 629, 36500 Irapuato Gto, México;(2) Departamento de Ingeniería genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, Apartado postal 629, 36500 Irapuato Gto, México;(3) Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, Apartado postal 629, 36500 Irapuato Gto, México |
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Abstract: | 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 μmol mg−1 min−1, K
m
for ATP: 67 μM, K
m
for syntide 2: 15 μM). The kinase showed an optimum pH of 6.8 and a marked dependence on low-micromolar Ca2+ concentrations (K
d
: 0.77 μM). 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.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . |
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Keywords: | Beta vulgaris Calcium-dependent protein kinase Mass spectrometry Plasma membrane |
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