The overall goal of this procedure is to quantitate the in vitro ATPase activity of purified proteins for functional characterization. This method can be used to characterize new putative ATPases, evaluate the effective potential activators or inhibitors and to determine the contribution of particular domains or residues to ATPase activity. The main advantage of this technique is that it is a simple, sensitive method for measuring in vitro ATPase activity and can be easily optimized.
Prepare stocks of all the necessary reagents for incubation with purified protein as instructed in the text protocol. Premix 100 millimolar magnesium chloride and 100 millimolar ATP at a one to one ratio just before setting up the ATP hydrolysis reaction. Prepare and label 1.5 milliliter tubes for collecting samples at regular intervals throughout the reaction.
Prepare tubes to collect samples at times zero as well as at times of 15, 30, 45 and 60 minutes. Add 245 microliters of 1x HNG buffer to each tube to dilute the samples collected from the ATP hydrolysis reactions at a dilution of one to 50. Next, prepare a bath of dry ice and ethanol for quickly freezing samples to stop the reaction.
In a rubber ice bucket or other safe container, add several piece of dry ice and carefully pour enough 70%to 100%ethanol to cover the dry ice. Dilute the purified protein in 1X HNG buffer and keep it on ice. Set up the ATP hydrolysis reactions in the prepared 0.5 milliliter tubes by adding a pre-calculated volume of water to reach a final reaction volume of 30 microliters.
Followed by six microliters of 5X HNG or other buffer, three microliters of 100 millimolar magnesium chloride ATP mixture and 0.25 to five micromolar protein. Incubate a buffer only negative control condition in which no protein is added. Next, remove five microliters from the reaction at times zero.
Then dilute the aliquot one to 50 in the prepared 1.5 milliliter tube containing 245 microliters of HNG buffer. Immediately freeze the sample in the dry ice ethanol bath. Incubate reactions at 37 degrees Celsius to allow ATP hydrolysis to occur for one hour.
At each interval remove five microliter aliquots from the reaction and add to labeled sample tubes containing HNG buffer as before. At the end of the ATP hydrolysis reaction, move diluted samples to a 80 degree Celsius freezer for storage. To ensure all samples are completely frozen, wait at least 10 minutes before proceeding.
Thaw the diluted samples containing ATP hydrolysis reaction aliquots from each time point at room temperature. Next, prepare a 96 well plate containing samples and phosphate standards. In a 0.5 milliliter tube dilute the phosphate standard from 800 micromolar to 40 micromolar by adding 5.5 microliters of the standard to 104.5 microliters of HNG buffer.
Mix well and add 100 microliters of this 40 micromolar phosphate standard to well A1 of the 96 well plate. Add 50 microliters of HNG buffer to wells B1 through H1 for one to one serial dilutions of the phosphate standard. Remove 50 microliters of 40 micromolar phosphate from well A1, add it to the 50 microliters of assay buffer in well B1 and mix.
Then remove 50 microliters from well B1 and add it to well C1, continuing dilutions through well G1.Discard 50 microliters from well G1 after mixing to ensure each well has the same volume. Well H1 should contain only buffer to create a phosphate standard from 40 to zero micromolar. Next, add 50 microliters of each sample in duplicate to the plate.
Add samples from the same time point in columns vertically and from different time points horizontally. This allows for up to eight samples and five time points per plate. Use a sterile pipette to remove enough malachite green meliptate phosphate detection reagent to add 100 microliters to each of the wells containing samples and standards.
Add the detection reagent to a dish for easy pipetting using a multi-channel pipette. Do not pour the detection reagent directly into the dish as phosphate contamination is likely to occur. Using a multi-channel pipette, add 100 microliters of the phosphate detection reagent to each well and mix carefully by pipetting up and down a consistent number of times without introducing bubbles, working in order from the last time point to the first time point.
Leave the plate at room temperature for 25 minutes or according to the manufacturer's directions. To quantitate the results, read the absorbance of the samples at 650 nanometers using an absorbance micro plate reader. Representative results of kinetic and endpoint ATPases are shown, in which the activity of wild type and mutant forms of the type two secretion ATPase/EPSE is determined in the presence and absence of a stimulant cardiolipin.
Shown here are the results of a kinetic cardiolipin-stimulated ATPase, demonstrating the linear phosphate release over time for wild type EPSE and a double lysine mutant, with bovine serum albumin serving as a negative control. These data can be represented as the amount of phosphate released per minute divided by the protein concentration in order to quantitate and compare the ATPase activity of each protein. The data indicate that two lysine residues, K417 and K419, contribute to the cardiolipin-stimulated ATPase activity of EPSE.
Of comparison of the ATPase activity of the same proteins without cardiolipin-stimulation shows that these two lysine residues do not contribute to the low basal activity of EPSE, but rather to the ability of the protein to be stimulated by cardiolipin. After watching this video you should have a good understanding of how to quickly and accurately quantitate the ATPase activity of purified proteins. While attempting this procedure it is important to consider the sensitivity of the phosphate detection reagent.
To avoid phosphate contamination, we recommend using disposable plastic-ware and ultra-pure water and performing size exclusion or ion exchange chromatography following protein purification.
