How to estimate the activity of liver glucose 6 phoshatase and phosphoenol pyruvate carboxy kinase (PEPCK) by spectroscopic method?
Hi! Maybe you can conjugate glucose with some fluorophore, for example glucose-bodipy and you can see how is processed and also maybe you can conjugate to other molecule of your interes!
I'm not sure how well these ideas will work with a liver homogenate, but here are some examples of simple spectrophotometric methods that can be used to assay the activity of these enzymes. The reagents for these methods are commercially available.
The products of glucose-6-phosphatase are glucose and inorganic phosphate.
Inorganic phosphate can be detected in an endpoint absorbance assay using a Malachite Green/molybdate reagent. It can also be detected in a continuous absorbance assay using purine nucleoside phosphorylase as a coupling enzyme and 2-amino-6-mercapto-7-methylpurine riboside (MESG) as the detection reagent. Here is information from a kit for this assay: https://tools.thermofisher.com/content/sfs/manuals/mp06646.pdf.
The question is whether the background level of inorganic phosphate in the extract is low enough to allow these assays to be used.
Glucose can be detected using glucose oxidase and peroxidase coupling enzymes and Amplex Red as the detection reagent (one of many methods). Here is a kit for this purpose: https://www.thermofisher.com/order/catalog/product/A22189.
Again, the utility of glucose detection depends on the background level of glucose in the extract being low enough. You may find it necessary to dialyze the extract to reduce the background levels of phosphate and glucose.
Phosphoenolpyruvate carboxykinase (PEPCK) converts oxaloacetate into phosphoenolpyruvate (PEP) and carbon dioxide. PEP can be detected using pyruvate kinase (PK), lactate dehydrogenase (LDH), ADP and NADH to make pyruvate, ATP, and NAD. Reaction is followed continuously by the decrease in NADH absorbance at 340 nm. PK and LDH can be purchased as a premix.
Here is a protocol for a different PEPCK assay that follows the reverse reaction:
http://home.sandiego.edu/~josephprovo/pepck%20assay%20and%20homogenization.pdf
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry