I am looking to measure enzymatic activity of the glycolytic pathway by measuring ATP production over time. Most ready made kits on the market provide single time point measurments and do not seem to allow time-course measurements. Does anyone know of a way to do this?
I think that I have come across with a researcher that used luciferase to measure ATP formation, but i cannot remember the exact protocol, nor if it was a kinetics measure or end-point. Maybe with a little research, you may come across some infomation that describes that.
Most protocols in the literature use luciferase as an end-point measure unfortunately. I was wondering if it might be possible to monitor its activity over time in a combined enzymatic reaction (glycolytic enzymes produce ATP and ATP is used as substrate by luciferase that produces light). I know that this technique is often used with enzymes that use or produce NADH in order to shift the wavelength measurement from UV to visible light.
A difficulty with monitoring ATP production continuously in the entire glycolytic pathway is that such a method would have to consume (or at least bind up) the ATP, and ATP is required for the early steps in the pathway.
In addition to net production of ATP, the glycolytic pathway also produces NADH. You could follow the NADH production continuously by using the enzyme diaphorase combined with XTT. Diaphorase and NADH reduce XTT to produce a colored product. Of course, this only follows glycolysis through glyceraldehyde-3-phosphate dehydrogenase, before ATP production. Since NADH is not required for downstream steps, and NAD is regenerated, monitoring NADH production does not perturb the rest of the pathway.
An ideal method would detect the final product, pyruvate, without perturbing the rest of the pathway. There are a lot of enzymes that use pyruvate as a substrate, so perhaps you could come up with a coupled enzyme system to detect it. You can search for pyruvate-using enzymes at brenda-enzymes.org
Thank you for your suggestions. Indeed I had thought of NADH, but as you mentioned, I would only be able to measure the product of one step of the pathway and I would be blind to whatever might be happening downstream.
For the final product of glycolysis, I know that LDH is present in my medium and therefore susceptible to use pyruvate as a substrate. I was wondering if there might be a way of monitoring lactate and pyruvate quantities over time or at given time points, if ATP is too complicated to monitor.
If there is a sufficient amount of LDH present, it will consume the pyruvate to make lactate and return the NADH to NAD. The net effect will be no change in NAD concentration and no production of NADH, hence no signal in the diaphorase/XTT assay. That leaves you with measuring the accumulation of lactate. You might be able to detect its formation continuously using a lactate oxidase enzyme, combined with horseradish peroxidase and Amplex Red for detection of the hydrogen peroxide product. These materials are commercially available. If you decide to take that approach, you should also add more LDH to make sure it is not rate limiting. LDH is also commercially available.
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