Determination of picomole amounts of glycerate 3-phosphate, glycerate 2-phosphate, and phosphoenol pyruvate by an enzymatic assay coupled to firefly luciferase/luciferin luminescence.
A procedure for the determination of picomole amounts of glycerate 3-phosphate, glycerate 2-phosphate, and phosphoenol pyruvate is described. These metabolites were utilized by the glycolytic enzymes phosphoglycerate mutase, enolase, and pyruvate kinase to generate ATP which was determined by firefly luciferase/luciferin luminescence. The phosphoglycerate mutase used was of the glycerate 2,3-bisphosphate-independent type and was prepared from wheat germ. Stoichiometric conversion of glycerate 3-P, ranging in amount from 9 to 275 pmol, occurred after 25 min preincubation and required a narrow range of added mutase. The application of the procedure for determining these metabolites in suspensions of plant protoplasts is described.
D000244:Adenosine Diphosphate / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis; D049454:Firefly Luciferin; D005988:Glyceric Acids / Q000032:analysis*; D008156:Luciferases; D008163:Luminescent Measurements; D008832:Microchemistry; D010728:Phosphoenolpyruvate / Q000032:analysis*; D010736:Phosphoglycerate Mutase; D010936:Plant Extracts / Q000032:analysis; D014908:Triticum / Q000201:enzymology
D005988:Glyceric Acids; D010936:Plant Extracts; C008885:2-phosphoglycerate; D049454:Firefly Luciferin; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; C005156:3-phosphoglycerate; D000255:Adenosine Triphosphate; D008156:Luciferases; D010736:Phosphoglycerate Mutase