Measuring In Vitro ATPase Activity for Enzymatic Characterization

Summary

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Abstract

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Introduction

ATPases are integral enzymes in many processes across all kingdoms of life. ATPases act as molecular motors that use the energy of ATP hydrolysis to power such diverse reactions as protein trafficking, unfolding, and assembly; replication and transcription; cellular metabolism; muscle movement; cell motility; and ion pumping^1-3. Some ATPases are transmembrane proteins involved in transporting solutes across membranes, others are cytoplasmic and may be associated with a biological membrane such as the plasma membrane or those of organelles.

AAA+ ATPases (ATPases associated with various cellular activities) make up a large group of ATPases that share some sequence and structural conservation. These proteins contain conserved nucleotide binding motifs such as Walker-A and -B boxes and form oligomers (generally hexamers) in their active state¹. Large conformational changes in these proteins upon nucleotide binding have been characterized among diverse members of the AAA+ family. EpsE is a AAA+ ATPase and member of the bacterial Type II/IV secretion subfamily of NTPases^4-6. EpsE powers Type II Secretion (T2S) in Vibrio cholerae, the causative agent of cholera. The T2S system is responsible for the secretion of a wide variety of proteins, such as the virulence factor cholera toxin that causes profuse watery diarrhea when V. cholerae colonizes the human small intestine⁷.

Techniques for quantitating in vitro ATPase activity are varied, but commonly measure phosphate release using colorimetric, fluorescent, or radioactive substrates^8-11. We describe a basic method for determining in vitro ATPase activity of purified proteins using a colorimetric assay based on a commercially available malachite green-containing substrate that measures liberated inorganic phosphate (Pi). At low pH, malachite green molybdate forms a complex with Pi and the level of complex formation can be measured at 650 nm. This simple and sensitive assay may be used to functionally characterize new ATPases and to evaluate the roles of potential activators or inhibitors, to determine the importance of domains and/or specific residues, or to assess the effect of particular treatments on enzymatic activity.

Protocol

1. Perform ATP Hydrolysis Reaction with Purified Protein

1. Prepare Stocks of All the Necessary Reagents for Incubation with Purified Protein.
   1. Prepare 5x HEPES/NaCl/glycerol (HNG) buffer containing 100 mM HEPES pH 8.5, 65 mM NaCl, and 5% glycerol (or other assay buffer as appropriate).
   2. Prepare 100 mM MgCl₂ (or other metal, if ATPase is metal-dependent) in water.
   3. Prepare fresh 100 mM ATP in 200 mM Tris Base (do not adjust pH further) using high purity ATP. Aliquot and store the ATP stock at -20 °C for no longer than a few weeks, as ATP will break down over time, and refrain from freezing and thawing the ATP stock.
   4. Premix MgCl₂ and ATP at a 1:1 ratio just before setting up the ATP hydrolysis reaction.
2. Prepare and label 1.5 ml tubes for collecting samples at regular intervals throughout the reaction. Prepare tubes to collect samples at time 0 and at time 15, 30, 45, and 60 min.
   NOTE: As an alternative, collect samples only at time 0 and the endpoint.
   1. Add 245 µl 1x HNG buffer to each tube in order to dilute samples collected from the ATP hydrolysis reactions 1:50.
3. Prepare a bath of dry ice and ethanol for quickly freezing samples to stop the reaction. In a rubber ice bucket or other safe (non-plastic) container, add several pieces of dry ice and carefully pour enough 70-100% ethanol to cover the dry ice.
4. Dilute purified protein in 1x HNG buffer, as appropriate (typically 5-10 µM), and keep on ice.
5. Set up the ATP hydrolysis reactions.
   1. In separate 0.5 ml tubes for each sample, add the following reagents (in order): H₂O (up to a final volume of 30 µl), 6 µl 5x HNG or other buffer, 3 µl 100 mM MgCl₂-ATP mixture, and 0.25-5 µM protein.
   2. Include a buffer-only negative control condition in which no protein is added.
6. Remove 5 µl from the reaction at time 0, dilute 1:50 in the prepared 1.5 ml tube containing 245 µl HNG buffer, and immediately freeze the sample in the dry ice/ethanol bath.
7. Incubate reactions at 37 °C to allow ATP hydrolysis to occur for 1 hr. At each interval (15, 30, 45, and 60 min), remove 5 µl aliquots from the reaction and add to labeled sample tubes containing HNG buffer as in step 1.6.
8. At the end of the ATP hydrolysis reaction, move diluted samples to a -80 °C freezer for storage. To ensure all samples are completely frozen, wait at least 10 min before proceeding.

2. Incubate Samples Containing Free Pi with Detection Reagent

1. Thaw diluted samples containing ATP hydrolysis reaction aliquots at each time point (obtained from steps 1.6 and 1.7) at room temperature.
2. Set up a 96-well plate containing samples and phosphate standards.
   1. In a 0.5 ml tube, dilute the phosphate standard (provided with Pi detection reagent) from 800 µM to 40 µM by adding 5.5 µl of the 800 µM standard to 104.5 µl HNG buffer. Mix well, and add 100 µl of this 40 µM Pi standard to well A1 of a 96-well plate.
   2. Add 50 µl HNG buffer to wells B1-H1 for 1:1 serial dilutions of the Pi standard.
   3. Remove 50 µl of 40 µM Pi from well A1 and add to 50 µl assay buffer in well B1, mix, and remove 50 µl from well B1 and add to well C1, continuing dilutions through well G1. Discard 50 µl from well G1 after mixing to ensure each well has the same volume. Well H1 should contain only buffer to create a Pi standard from 40 to 0 µM.
      NOTE: If an additional factor (such as an inhibitor) has been added to the samples, create a standard curve containing that factor to control for changes in phosphate release or absorbance under those conditions.
   4. Add 50 µl of each sample in duplicate to the plate. Add samples from the same time point in columns vertically (sample 1 time 0 = A2, A3; sample 2 time 0 = B2, B3) and different time points horizontally. This allows for up to 8 samples and 5 time points per plate.
3. Use a sterile pipet to remove enough malachite green/molybdate Pi detection reagent to add 100 µl to each of the wells containing samples and standards (reagent needed (ml) = 0.1 x number of samples and standards) and add to a dish for easy pipetting using a multichannel pipet. Do not pour the detection reagent directly into the dish, as Pi contamination is likely to occur.
4. Using a multichannel pipet, add 100 µl of the Pi detection reagent to each well and mix by carefully pipetting up and down a consistent number of times without introducing bubbles, preferably in order from the last time points to the first time points.
5. Incubate the plate for 25 min at room temperature, or according to the manufacturer's directions.

3. Quantitate Results Using a Microplate Reader

1. Read the absorbance of the samples at 650 nm using an absorbance microplate reader.
2. Make a Pi standard curve. Using a graphing software, graph the absorbance values for the Pi standard samples versus concentration in order to find an equation used to solve for the amount of phosphate in each sample.
3. Calculate the ATPase activity for each sample by calibrating with the phosphate standard. Phosphate released = (OD₆₅₀ - Y intercept)/slope.
   1. Average the total Pi from duplicates of each sample. Subtract the buffer-only control's absorbance reading from this number. Multiply this by the dilution factor (50 in our example).
   2. Determine the nmol Pi released per µmol protein. Graphing these values for each time point in a kinetic assay should yield linear fits of at least R = 0.99 (Figure 1); If not, the assay may be adjusted with more or less protein or a longer or shorter incubation time.
4. Represent the results as nmol Pi/µmol protein/min (Figures 2, 3), or as nmol Pi/µg protein/min if desired.

Results

The in vitro activity of the T2S ATPase EpsE can be stimulated by copurification of EpsE with the cytoplasmic domain of EpsL (EpsE-cytoEpsL) and addition of the acidic phospholipid cardiolipin¹². It is also possible to determine the role of particular EpsE residues in ATP hydrolysis by comparing activity of wild type (WT) to variant forms of the protein using this assay. Here, the effect of substituting two lysine residues in the EpsE zinc-binding domain is measured by...

Discussion

This is a general protocol for measuring in vitro ATPase activity of purified proteins for biochemical characterization. This method is easily optimized; for example, adjusting the amount of protein, buffer and salt compositions, temperature, and varying the assay length and intervals (including increasing the total number of intervals) can improve activity quantitation. Commercially available malachite green-based reagents are highly sensitive, and can detect small amounts of free phosphate (~50 pmol in 100
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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HEPES buffer	Fisher	BP310-500
Sodium chloride	Fisher	BP358-212
Magnesium chloride	Fisher	BP214-500
Adenosine triphosphate (ATP)	Fisher	BP41325
96-well plates (clear, flat-bottom)	VWR	82050-760
BIOMOL Green	Enzo Life Sciences	BML-AK111	Preferred phosphate detection reagent. Caution: irritant.
Microplate reader			BioTek Synergy or comparable
Prism 5	GraphPad Software