Ca2+-dependent protein kinase from alfalfa (Medicago varia): Partial purification and autophosphorylation |
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| Institution: | 1. National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, People''s Republic of China;2. I3Bs Research Institute on Biomaterials, Biodegrabilities, and Biomimetics, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of Minho, Barco 4805017, Portugal;1. Centre for Microbiology and Environmental Systems Science, Department of Environmental Geosciences, University of Vienna, Althanstrasse 14 UZAII, 1090 Vienna, Austria;2. Geological Survey of Spain (IGME-CSIC), C/Ríos Rosas 23, 28003 Madrid, Spain;3. Agriculture, Nautical, Civil and Maritime Engineering Department. University of La Laguna (ULL), Ctra. Geneto, 2, La Laguna, 38200 Tenerife, Canary Islands, Spain |
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| Abstract: | A calcium-dependent protein kinase (CDPK) was purified to 1400-fold from the soluble fraction of alfalfa (Medicago varia) cells by ammonium sulfate fractionation, Sephacryl-300, DEAE-Sephacel, Phenyl-Sepharose and Hydroxylapatite column chromatography. The enzyme is mainly monomeric. During the course of the purification steps a 50 kDa phosphoprotein doublet and a 56 kDa phosphoprotein copurified with the CDPK activity. Mobility shift of these proteins have been shown by SDS PAGE in Ca2+ free conditions. Tests on enzyme activity after separation by native gel electrophoresis revealed two protein kinase activities in our enzyme preparation and the phosphorylation of the 50 kDa and 56 kDa proteins. We suggest that these proteins are the autophosphorylated forms of calcium dependent protein kinases. Preincubation of the CDPK in ATP resulted in a marked increase in enzyme activity, but did not alter the Ca2+ sensitivity of the protein kinase. |
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