Assaying for pyruvate,orthophosphate dikinase activity: Necessary precautions with phosphoenolpyruvate carboxylase as coupling enzyme |
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| Authors: | G Salahas Y Manetas N A Gavalas |
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| Institution: | (1) Laboratory of Plant Physiology, Department of Biology, University of Patras, Patras, Greece |
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| Abstract: | Phosphoenolpyruvate carboxylase (EC 4.1.1.31), used as a coupling enzyme in the assay of the pyruvate, orthophosphate dikinase (EC 2.7.9.1) forward reaction, is a serious limiting factor for the overall rate when added at a level of 0.2–0.3 unit/ml of assay medium. Nonlimiting assay conditions are obtained by either increasing the level of the coupling enzyme to 3 units/ml or adding 6mM glucose-6-phosphate as an activator/stabilizer of phosphoenolpyruvate carboxylase.Abbreviations G-6-P
glucose-6-phosphate
- LDH
lactate dehydrogenase
- MDH
malate dehydrogenase
- PEP
phosphoenolpyruvate
- PEPCase
phosphoenolpyruvate carboxylase
- PVP
polyvinylpyrrolidone
- PPDK
pyruvate, orthophosphate dikinase
- U
unit of enzyme activity ( mol/min) |
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| Keywords: | assaying technique glucose-6-phosphate phosphoenolpyruvate carboxylase pyruvate Pi (orthophosphate) dikinase |
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