Hello all,
I'm currently working with undergraduate student-researchers (there are no graduate students at my university) to purify and characterize the drug targetability of glucokinase from pig liver. We thought it would be a fairly feasible project considering the limited hours that student-researchers can invest (given their abundant courseloads) and the limited infrastructure in a primarily-undergraduate institution.
After several weeks of lab work, it would appear that what we thought was glucokinase activity may in fact be another enzyme. The enzyme activity seems to run perfectly fine (even better, in fact) despite omitting ATP from the assay below:
Glucose + ATP --> glucose-6-phosphate + ADP
glucose-6-phosphate + NADP+ --> 6-phosphoglucono-delta-lactone + NADPH + H+
I've wracked my brain trying to figure out why the enzyme activity would be as high (or higher!) in the absence of ATP in the enzyme assay, and the only conclusion I can come up with is that we're actually somehow assaying glucose dehydrogenase, with a reaction of glucose + NADP+ --> glucono-delta-lactone + NADPH + H+.
Has anyone encountered this conundrum and been able to resolve it?
Before you answer, allow me to please add the following:
- We do NOT have access to SDS-PAGE methodologies to determine the molecular weight of the enzyme in question; in fact, we haven't gotten significantly far in our purification beyond a resuspension of the 40% ammonium sulfate-precipitated pellet.
- We do NOT have access to the resources and consumables needed to express and purify recombinant proteins -- hence why we're working with pig livers.
https://www.sciencedirect.com/topics/nursing-and-health-professions/glucose-dehydrogenase
check this link
A strategy you could try to help with the purification, which would require nothing other than what you already have, would be to measure the activity with and without ATP. The difference between those measurements better represents the glucokinase activity, which you could then use to track the purification.
Adam B Shapiro Thanks so much for the answer Adam. I'll certainly do that. What is seemingly paradoxical about that, is that the activity is higher without ATP than with ATP. I'm confounded as to what that means.
I agree. If the enzyme activity is higher in the abscence of ATP then it isn't Glucokinase and you might be assaying the product of some other reaction. Assuming you have isolated Glucokinase as expected, are you confident about your asssay conditions ? Glucokinase activity is directly proportional to the supply of glucose. It requires about 4 to 10 mM of glucose and about 100 mM of a magesium salt to change its conformation and have optimum activity. And the pH needs to be around pH 8.5-8.7. I apologize if this seems rudimentary but I cant stress how many assays I have botched up by not being careful with the pH. If you still dont see a change, then the ammonium sulphate cut didnt preciptate glucokinase and you may need to redo it with controls as suggested.
What ATP concentration are you using? Some ATP is supplied as an incompletely neutralized salt (e.g. disodium), resulting in an acidic solution. If the ATP concentration was high enough and the buffer wasn't very strong, it could have lowered the pH significantly.
I'd like to thank everyone for their answers and help. After a few more experiments, I can more confidently say that it does seem very likely that we have two enzyme activities that are "interfering" with each other, for lack of a better term: hepatic glucokinase (dependent on both glucose and ATP) and a very-unexpected hepatic glucose dehydrogenase activity.
We need to resolve the two enzymes from one another -- of course, this is easier said that done, trying to get undergraduates into the lab long enough to carry out extensive protein purification.
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