I am working with a protein that uses GTP and ATP as substrates and form hyperphosphorylated GTP and AMP as products. So to monitor the reaction I want an assay, (other than radioactive assay) where I can quantify either of the remaining amount of substrates or the products formed. Since both substrates and products are nucleotides what could be the better assay? any suggestion.....
Since fluorescent analogs have bulky substitutions that might not give true affinity of proteins for substrate, so any analog that has no substitution but still can be detected by fluorescence or luminescence.
You may consider a HPLC method like that described in the following paper:
Micheli, V., Sestini, S., Rocchigiani, M., Jacomelli, G, Manzoni, F., Peruzzi, L., Gathof, B.S., Zammarchi, E., Pompucci, G., 1999. Hypoxanthine-guanine phosphoribosyltransferase deficiency and erythrocyte synthesis of pyridine coenzymes. Life Sci. 64, 2479-2487. DOI: 10.1016/S0024-3205(99)00205-2
A high throughput method for detecting AMP uses 3 commercially available coupling enzymes and substrates as follows:
adenylate kinase (also called myokinase): AMP + ATP 2 ADP
pyruvate kinase: ADP + phosphoenolpyruvate ATP + pyruvate
lactic dehydrogenase: NADH + pyruvate NAD + lactate
The reaction is followed by the decrease in NADH absorbance at 340 nm.
The pyruvate kinase and lactic dehydrogenase can be purchased as a premix from Sigma.
Agree with Adam.With these coupled enzymes you can design a continuous method, that must of course be validated testing if the reaction velocity depends linearly on the concentration of your protein. You must test this at the lowest and at the highest substrate (GTP and ATP) concentrations. You must add the three coupled enzymes and cosubstrates (phosphoenolpyruvate) in amounts such that they are not rate-limiting. IAn extra advantage of a continuos method based in the use of these three coupled enzymes, is that the [ATP] will be kept constant during the reaction, since it is regenerated by the coupled system. If the ATP concentrations you used are low, I presumed that the amount of myokinase will be crucial.
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