Hi Bhaskar

I would suggest to follow this paper...

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078443

There are many ATPase assays available, including the 2 you mentioned.

PK/LDH,which measures ADP, is relatively insensitive, but has the advantage that it is continuous, which allows you to measure low amounts of ADP production by using the slope of the progress curve, and is compatible with high ATP concentrations. A drawback is interference caused by compound absorbance at 340 nm.

The Malachite Green (MG) assay, which measures phosphate, can be sensitive in the single-digit micromolar range. It is an endpoint assay. It can suffer from interference by compounds that interact with the dye. It can have trouble with high ATP concentrations because of background levels of phosphate and because the acidity of the reagent causes spontaneous ATP hydrolysis.

There are also many commercial ADP detection reagents, although they can be expensive.

If you have to use a high ATP concentration (hundreds of micromolar to mM), PK/LDH is probably a better choice than MG. Note that ADP contamination in your ATP will give a signal, but this can be dealt with by adding the ATP to the PK/LDH/PEP/NADH reagent before adding the enzyme, so that the ADP gets converted back to ATP before the start. Monitor the enzyme reaction continuously, and use the slope of the linear initial part of the reaction. Use the largest volume in the assay plate you can to get the largest slope. The NADH concentration determines the initial absorbance, which should not exceed 1. Use 384-well plates, if possible, to minimize the volume of reagents needed, and make sure the samples in the plate are well-mixed. Use a good absorbance plate reader that has a noise level of 0.0001 absorbance unit.

Hi Adam,  your answer is very informative. I am doing some GTPase measurement recently using PK/LDH system.  But one thing I wonder is whether the commercial PK/LDH enzyme could have any GDP/GTP contamination? My NADH signal always decays linearly even without adding GTP. I tried to repurify and ultrafilter my protein many time which doesn't help. The only thing helped is to use less PK/LDH.

The decrease in NADH signal with time in the absence of GDP could just be due to degradation of the NADH by itself. Have you measured the signal decrease when PK/LDH is omitted? If reducing the PK/LDH concentration alleviates the problem, can you reduce it while still having enough that the PK/LDH reaction is not rate-limiting?

Thanks Adam. Yes, I tried to test the decay without PK/LDH as you mentioned. It does decrease, but in a ~0.005 absorbane/min scale. After adding my GTPase, FtsZ, this rate increased to ~0.06 absorbane/min. I will try to use as small amount as I can. But does this ever happen to you? And I noticed different PK/LDH batches from Sigma also showed different background rate in my system.

Your system contains FtsZ (a GTPase), GTP, NADH, PEP, and PK/LDH.

If there is any GDP present in the PK/LDH, NADH will be converted to NAD and the absorbance will decrease when no FtsZ or GTP is present. Once the GDP is used up (converted to GTP), the absorbance decrease will stop. Therefore, you could preincubate the PK/LDH, PEP, and NADH until the absorbance decrease stops. Then start the FtsZ reaction by adding GTP (free of GDP, if possible) and FtsZ sequentially.

If your GTP is contaminated with GDP, you could include the GTP in the preincubation with PEP, NADH, and PK/LDH and wait for the absorbance decrease to stop, then initiate the FtsZ reaction by adding FtsZ.

Thanks for you help again, Adam. It worked by using a very small amount of PK/LDH!

 I´ve been working with several sources of Na,K-ATPase during the past 20 years, and in my opinion, the best method to evaluate hydrolytic ATP activity is the radioactive assay employing 32Pi-ATP....My protocol is based on Grubmeyer´s and Pennnefski, 1983 paper which extracts 32Pi employing the addition of activated charcoal in acidic media to pellet the organic matter of the assay. Then, after a benchtop centrifugation step, the resulting supernatant is counted with LSC techniques. The advantages: it can be performed with very low samples of the purified or non purified enzyme (as low as 5-10 uG) - Easy to count if you have a LSC system with a scintillator based on toluene+PPO+POPOP which are very cheap in comparison with luminiscent reagents...Very much more sensitive than coupled assays as PK/LDH system! We actually measure the Na,K-ATPase from the gills of euryhaline crab C. danae employing this method with very much reliable results.

I am working on N-acetyl glucosamine kinase (NAGK) from insects, recombinantly expressed and purified. I am trying to do a coupled-enzyme assay for NAGK. However, I get a steady decline in absorbance in my blank reaction (devoid of NAGK), consisting of 100 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 4 mM ATP, various concentrations of GlcNAc, 0.2 mM NADH, 1 mM phosphoenol pyruvate, 10 units of pyruvate kinase/lactate dehydrogenase. Initially, I thought it is because of ADP contamination along with ATP, and hence I waited until the blank to reach equilibrium and after then added NAGK. This definitely triggered the reaction as there was again decline in the absorbance at 340 nm but there is no change in the rate of reaction with respect to concentrations of substrate GlcNAc. However, in few instances I did observed some changes in the rate of reaction but there was no uniformity in the outcomes and the results have never really been in accordance with the Michaelis-Menten curve. I have repeated the assays several times but could not find any satisfactory outcome and I am not able to trace my mistake till now.

Kindly suggest me the needful to solve this issue. It will be of great help for me.

Thanks in advance.

Xinxing Yang How much units of PK/LDH you had added for each assay? Did it solve the issue of absorbance in your blank samples?

You need to have some potassium in the assay for the activity of the pyruvate kinase.Another problem may be that the PK and/or LDH concentration is not high enough, so that the NAGK reaction is not the rate-limiting step, as it should be. Another possibility is that all your GlcNAc concentrations are much higher than the Km, so all the rates are the same (Vmax).

Thanks Adam For your valuable suggestion. I am using premixed PK/LDH (sigma P0294) which is buffered with 100 mM KCl. I have tried upto 30 units/ml PK/LDH but it yielded same results. Many a times it happened that there is no change in absorbance when GlcNAc and NAGK are added after preincubation of pk/ldh, pep, atp and mgcl2 in 100mM Tris buffer. However, it showed steep decline in absorbance with same protocol. This again create doubts whether NAGK is working or not!