Phosphoenolpyruvate carboxykinase from higher plants: Purification from cucumber and evidence of rapid proteolytic cleavage in extracts from a range of plant tissues |
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Authors: | Robert P Walker Stephen J Trevanion Richard C Leegood |
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Institution: | (1) Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield, S10 2UQ Sheffield, UK |
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Abstract: | Phosphoenolpyruvate carboxykinase (PEPCK) was purified 600-fold to homogeneity from the cotyledons of cucumber (Cucumis sativus L.) and a polyclonal antiserum raised. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the purified preparation contained a single polypeptide of 62 kDa, consistent with previous studies of this enzyme in C4 grasses. Immunoblots of crude extracts showed that a form of PEPCK of approximately this molecular mass predominated in cucumber cotyledons and in a range of plant tissues (cotyledons of fat-storing seedlings, leaves of C4 and Crassulacean acid metabolism plants). However, when these tissues were extracted in the presence of SDS and the extracts analysed by immunoblotting, a larger polypeptide of 68–77 kDa was detected. Thus the enzyme generally measured in crude extracts is a smaller form which arises by rapid proteolysis. This phenomenon means that the native form of PEPCK has never been purified from plants nor its properties determined.Abbreviations CAM
Crassulacean acid metabolism
- DTT
dithiothreitol
- PEG
polyethyleneglycol
- PEP
phosphoenolpyruvate
- PEPCK
phosphoenolpyruvate carboxykinase
- Rubisco
ribulose-1,5-bisphosphate carboxylase-oxygenase
We are grateful to Dr. Steve Smith (University of Edinburgh, UK) for helpful discussions, Dr. Alf Keys (Institute of Arable Crops Research, Rothamsted, UK) for the gift of pure Rubisco and Dr. Tristan Dyer (John Innes Centre for Plant Science Research, Norwich, UK) for the antiserum to fructose-1,6-bisphosphatase. This research was supported by the joint Agricultural and Food Research Council/Science and Engineering Research Council Programme on the Biochemistry of Metabolic Regulation in Plants (PG50/590). |
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Keywords: | C4 plant Crassulacean acid metabolism Cucumis Phosphoenolpyruvate carboxykinase (Purification) Proteolytic degradation |
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