Fructose bisphosphatase (EC 3.1.3.11) is an enzyme that converts fructose-1,6-bisphosphate to fructose 6-phosphate in gluconeogenesis and the Calvin cycle which are both anabolic pathways. I need a real method to measure it in liver homogenate.
Here is a method from yeast extract:
Fructose bis phosphatase makes fructose 6 phosphate. Fructose 6 phosphate is converted to glucose6 phosphate by phosphoglucoseisomerase. Then glucose 6 phosphate dehydrogenase is used to oxidize glucose 6 phosphate and convert NADP to NADPH. The reaction is followed at 340nm.
Could you please support me with full protocol "reagents, procedure and calculation" that would be greatly appreciated.
Enzymatic assay:
In 1 ml (spectrophotometer cuvettes)
Mixture Reaction 0.5 ml
(Imidazol 100 mM, pH 7.1; KCl 200 mM, EDTA 2mM, MgSO4 10 mM)
PGI/G6PDH (diluted 50 fold in water) 0.05 ml Crude extract 0.02 µl
NADP 10 mM 0.05 ml
Water until 0.95 ml
Read OD340 until stabilization
Add Fru1,6P2 5 mM 0.05 ml
Follow increase OD340 nm for 5 -15 min
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