The advantage of the pyrovate kinase/lactatedehydrogenase assay (Schwartz et al., 1971, Meth. Pharmacol. 1:361-388) is that by following spectroscopivally the consumption of NADH due to the exact stoichiometry of 1 NADH per 1 ATP hydrolyzed, you immediately can calculate the enzyme activity by a single equation. You do it from the slope of the absorbance decrease before and after addition of ouabain, and the difference is the enzyme activity of the Na,K-ATPase. With a computerized spectrometer you get the activity instantaneously. The disadvantage of the method ist that you have the pyrovate kinase, lactatedehydrogenase, PEP, and NADH in the same buffer as the Na,K-ATPase which you measure. This limits the choice of buffer composition, i.e. the pH range and ion composition and ion strength. You have to meet the needs of those both enzymes, and that restricts your choices, i.e. experiments under unphysiological conditions that are sometimes desired to test the pump under specific conditions.

The the case of the ammoniummolybdate (or Malachite-green test) you can choose any buffer condition or temperature, let the pump do its work for some time (typically many minutes or an hour), then stop the process (let's say on ice or by a stopping buffer). Then the test determines the amount of inorganic phosphate released by ATP hydrolysis. You get just one data point per incubation with the test reagents. Hence you do the phosphate determination always in triplicate to get a reliable result and its error. Phopshate background or contamination in the buffer or glassware are sometimes a problem. Therefore, each time you use this test, you have to perform in parallel a calibration "routine" with defined amounts of added phosphate in the expected concentration range. The absorbance vs. phosphate concentration dependence is fitted by a regression line and this line is used to determine the amount of phosphate released by the pump action from the measured absorbance. Again, you have to repeat the experiment also in the presence of ouabain to get a measure of some (unspecific) ATPase activity in your sample. The advantage of having no buffer restrictions stands against time and effort needed for the numerous absorbance measurements you have to perform to get your calibration and data points.

Therefore, we always prefer the pyrovate kinase/lactatedehydrogenase assay as long as the buffer conditions will allow.

Thank you for the eThank you for the extended answer it helps a lot. If I understand correctly the main difference is in the workload not the accuracy or detectionlimit. In the end I would like to extend this protocol to measure multiple atpase activities as done by Kong et al. “Seasonal variations of ATPase activity and antioxidant defenses in gills of the mud crab Scylla serrata (Crustacea, Decapoda)“.

At the moment I only tried the ammoniummolybdate protocol but I do not observe any difference between the measurements with and without ouabain. I do wander why the ‘contamination’ with phosphate is no problem in the pyrovate kinase/lactatedehydrogenase assay.

xtended answer

In the pyrovate kinase/lactatedehydrogenase assay phosphate is no substrate for any of the participating enzymes, PK requires PEP, LDH needs NADH, the sodium pump needs ATP and phosphate ihas there an inhibitory effect only at > mM.

If you don't see an effect of ouabain, this is suspicious. Either you don't have an active Na,K-ATPase present or its activity is so low that it is superposed by other ATPases in the membrane or by a high phosphate background.

Hans-Jürgen, have you ever tried to measure other atp-ases with this protocol? I assume that if you have a mixture with ouabain but with Ca, you measure only the remaining ATPase activity. If you then do the same without Ca you measure only the Mg ATPAse (and the difference should be the sum of Mg/Ca and Ca2+ atpase). Unfortunately removing Mg2+ from the reagent is not an option to split the latter two Atpases.

We worked with quite a number of P-type ATPases besides the Na,K-ATPase: SERCA, gastric H,K-ATPase, H-ATPase from Enterococcus hierae and the KdpFABC complex from E.coli. The PK/LDH assay was successfully and routinely used also for SERCA and the H-ATPase. For the H,K-ATPase we used it in the pH range 6 - 8. We worked preferentially with purified protein preps, in which other ATPases were present only as impurities. Hence we had no real problems. In the case of the Na,K-ATPase we prepared the enzyme from the out medulla of rabbit kidney in which the pump was present in high concentrations due to the physiological function of the cells (recovery of ions from the primary urine). The membrane preps we got after the first purification step ("microsomes") had an ouabain-inhibitable fraction of ATPase activity of ~50 %. After the second purification step it was >95 %. Therefore, we hadn't to fight to silence other ATPases (or to think about what kind of ATPases they were). Probably the dominant species was the Mg-ATPase. I'm not sure what kind of prep you use, but you must have access to the cytoplasmic side of the membranes otherwise you couldn't access to the ATP pool. But when you broke the cells, you might see also V- and F-type ATPasess from intracellular membranes. They can be inhibited with azide. You should try this by all means.

We worked with quite a number of P-type ATPases besides the Na,K-ATPase: SERCA, gastric H,K-ATPase, H-ATPase from Enterococcus hierae and the KdpFABC complex from E.coli. The PK/LDH assay was successfully and routinely used also for SERCA and the H-ATPase. For the H,K-ATPase we used it in the pH range 6 - 8. We worked preferentially with purified protein preps, in which other ATPases were present only as impurities. Hence we had no real problems. In the case of the Na,K-ATPase we prepared the enzyme from the out medulla of rabbit kidney in which the pump was present in high concentrations due to the physiological function of the cells (recovery of ions from the primary urine). The membrane preps we got after the first purification step ("microsomes") had an ouabain-inhibitable fraction of ATPase activity of ~50 %. After the second purification step it was >95 %. Therefore, we hadn't to fight to silence other ATPases (or to think about what kind of ATPases they were). Probably the dominant species was the Mg-ATPase. I'm not sure what kind of prep you use, but you must have access to the cytoplasmic side of the membranes otherwise you couldn't access to the ATP pool. But when you broke the cells, you might see also V- and F-type ATPasess from intracellular membranes. They can be inhibited with azide. You should try this by all means.

At the moment I work with a protocol described in "Na+/K+–ATPase activity and gill ultrastructure in the hyper-hypo-regulating crab Chasmagnathus granulatus acclimated to dilute, normal, and concentrated seawater. "

This is still based on ammoniummolybdate. No purification is performed at the moment (as done in several other papers). The buffer solution is a mixture of EDTA, NaCl, HEPES, PMSF. The incubation mixture consists of NaCl, MgCl, EDTA, imidazole, ATP and KCL (with or without ouabain).

There are others ways, the ATP P32, you used radioactivity, after I used the ouabain dependant and sodium Kpase acitivity. The coupled system with PK and LDH is dependant of detergent treated membranes, inside out and right side out vesicles.

As we can measure also the 3H-ouabain binding to Na,K-ATPase which is a measure of the sodium pump, you may have a calculation of the turnover rate of the enzyme (My paper in FEBS 1993. The problem is the characterization of your vesicles and to calculate the yield in enzymes between the homogenate fraction and the membrane purified fraction. For me, the best is a P2 fraction. Your yield is better than 50 % and I suppose that you will test an inhibition of the Na,K-ATPase, so a decrease of more than 50 % could not be attributed to a special membrane recovery. All that my research has be done with mammallian species. We have a research program with sea urchin.

Furthermore, the presence of isoenzymes of NKA is also important. You can use the kinetic analysis of the NKA. Another difficulties is the background ATPase. Heart with a big mitochondrial activity is a problem for the stability of the enzyme activity during the measure. If you are sure that the enzyme activity is linear over time, the molybdate method is intersting to have a point to point analysis for example between time O and One hour. The last point is the sodium, potassium affinities which differs among NKA isoforms.

To start a project, I have the same opinion that HJ Apell. The Kpase after with the real time spectrophotometric analysis is intersting.

Best regards

Both experimental protocols are applicable. Main disadvantage of ATP regenerating system (i.e., PEP, NADH, PK and LDH) using is that you have two other enzymes in system. Thus if you are looking for modulators of Na/K-ATPase activity, these substance may influence also activity of PK and LDH. This may induce problems in interpretation of results. If you are detecting activity of Na/K-ATPase as Na and K dependent and ouabain sensitive Pi liberation you will not have this problems. However, the concentration of ATP will decrease during ATPase reaction. This may affects estimation of ATPase activity. Therefore if you are using this protocol, you should control the linearity of Na/K-ATPase induced Pi liberation time dependence. For activity estimation you have to used only linear parts of this dependence.

ATPase activity can also be measured by a bioluminescent method (Lundin, A. and Eriksson, J. (2008) A real-time bioluminescent HTS method for measuring protein kinase activity influenced neither by ATP concentration nor by luciferase inhibition, Assay Drug Dev Technol 6 (4), 531-541). Although not stated in the title of the paper this is an excellent method for ATPase measurements. The ATP depletion can be continuously monitored in the presence of firefly luciferase. At low ATP concentrations (1 µmol/L; i.e. far below the Km) the depletion follows 1st order kinetics and the percentage ATP depletion can be calculated from dATP/dt=(ln(light at time t1)-ln(light at time t2)/(t1-t2). This value is independent of ATP concentrations far below the Km. Furthermore it is independent of luciferase activity as this cancels out in the calculation. Consequently the assay is extremely robust having a Z’-value of 0.96, which is extremely good. This property and the simple analytical procedure makes the assay very suitable for high throughput screening. I will be happy to provide technical support to anyone wishing to set up the assay.

ATPase activity can also be measured by a bioluminescent method (Lundin, A. and Eriksson, J. (2008) A real-time bioluminescent HTS method for measuring protein kinase activity influenced neither by ATP concentration nor by luciferase inhibition, Assay Drug Dev Technol 6 (4), 531-541). Although not stated in the title of the paper this is an excellent method for ATPase measurements. The ATP depletion can be continuously monitored in the presence of firefly luciferase. At low ATP concentrations (1 µmol/L; i.e. far below the Km) the depletion follows 1st order kinetics and the percentage ATP depletion can be calculated from dATP/dt=(ln(light at time t1)-ln(light at time t2)/(t1-t2). This value is independent of ATP concentrations far below the Km. Furthermore it is independent of luciferase activity as this cancels out in the calculation. Consequently the assay is extremely robust having a Z’-value of 0.96, which is extremely good. This property and the simple analytical procedure makes the assay very suitable for high throughput screening. I will be happy to provide technical support to anyone wishing to set up the assay.

I'll certainly look into this protocol, it indeed looks promising. I'm quite sure I will have plenty of questions but one question already did you check it at one wavelength of the total light emission?

In bioluminescence measurements one normally collects all the light emitted regardless of wavelength as this give maximum sensitivity. The specificity comes from the luciferase reaction being specific for ATP.

Determination of ATPase and Na,K-ATPase activity

ATPase and Na,K-ATPase activities were determined by a modification of the method of Post and Sen [5]. For enzyme reaction, 0.1 ml of 1000 mM sodium chloride–100 mM potassium chloride, 0.2 ml of 250 mM Tris–acetate buffer (pH 7.2), 0.1 ml of 1 mM EDTA, 0.1 ml of 30 mM magnesium chloride, 0.1 ml of 10 mM ouabain (or water), x ml of the enzyme source, and (0.3−x) ml of water, were put into glass tubes, and preincubated at 37°C for 5 min. Enzyme reaction was started by addition of 0.1 ml of 33.3 mM ATP, and the mixture was incubated at 37°C for 20 min. The enzyme reaction was stopped by addition of 2 ml of 1.5 M perchloric acid. After kept in ice-cold water for 20 min, the mixture was centrifuged (1700 g, 4°C, 10 min) to obtain the supernatant. Using the supernatant, the enzyme activity was determined by the two procedures mentioned below.

For colorimetric determination of Pi released from ATP, 0.5 ml of 4% sodium molybdate in 2.5 M sulfuric acid and 1 ml of 0.05% tin(II) chloride were added to 1 ml of the supernatant [6]. The mixture was kept at ambient temperature for 15 min, and absorbance was measured at 660 nm. Pi concentration was calculated from the regression line based on standard Pi solutions.

Hi David:

I tried with fish gill protocol of  McCormick, 1993 on invertebrate tissues and I found good response.  Good luck!

Dear David

 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 and 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..We actually measure the Na,K-ATPase from the gills of euryhaline crab C. danae employing this method with very much reliable results.  

Just in case...If you have the interest, I can share with you the detailed protocol we perform radioactive ATPase determinations......  

A cellular approach with real time resolution is presented in: Cell Metab. 2018 Nov 27. pii: S1550-4131(18)30682-X. doi: 10.1016/j.cmet.2018.11.005. [Epub ahead of print]

hello,

did anyone try to measure the Na-K-ATPase activity using an ELISA kit??

the kit i work with added that the unit to be used is U/L and i just thought that the ELISA measures the amount of protein not its activity