Abstract
Homogeneous phosphoribulokinase (PRK; ATP: d-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) was isolated from wheat leaves with a specific activity of 15 μkat mg-1 protein. The purification included ammonium sulfate cuts, isoelectric precipitation, and hydrophobic and affinity chromatography on pentylagarose and Blue Sepharose CL 6B, respectively. Gel filtration of the purified enzyme yielded a 83000 Da protein. Subunits of about 42000 Da were estimated from sodium dodecyl sulfate-polyacrylamide gels. Wheat leaf PRK was stable for at least four weeks when stored at 4°C. Saturation curves for ribulose 5-phosphate (Ru5P) and ATP followed Michaelis-Menten kinetics (K m values: K m Ru5P=50–80 μM; K m ATP=70 μM). The saturation curve for MgCl2 was sigmoidal (half-maximal velocity <0.5 mM). The affinity for Ru5P, ATP and Mg2+ was not affected by pH changes comparable to pH shifts in the stroma. In contrast to chloroplast fructose-bisphosphatase (Zimmermann et al. 1976, Eur. J. Biochem. 70, 361–367) the affinity for ligands remained unchanged in the dithiothreitol-activated and in the non-activated state. The activity of PRK was increasingly sensitive to inhibition by 3-phosphoglyceric acid with decreasing pH below pH 8.0.
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Abbreviations
- DTT:
-
dithiothreitol
- EDTA:
-
ethylenediamine-tetraacetic acid
- PRK:
-
phosphoribulokinase
- Ru5P:
-
ribulose-5-phosphate
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacryl-amide gel electrophoresis
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Surek, B., Heilbronn, A., Austen, A. et al. Purification and characterization of phosphoribulokinase from wheat leaves. Planta 165, 507–512 (1985). https://doi.org/10.1007/BF00398096
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DOI: https://doi.org/10.1007/BF00398096