CHAPTER TWENTY-FIVE
ATP and GTP Hydrolysis Assays (TLC) Vaishnavi Rajagopal, Jon R. Lorsch1 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Duration 4.2 Preparation 4.3 Note 4.4 Caution 5. Step 1 Steady-state ATP Hydrolysis 5.1 Overview 5.2 Duration 6. Step 2 Preparation of the TLC Plate and Chamber 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Tip 7. Step 3 TLC Separation of the Reaction 7.1 Overview 7.2 Duration 7.3 Tip 7.4 Tip 7.5 Tip References
326 327 327 328 329 329 329 329 329 330 330 330 331 331 331 331 331 332 332 332 332 333 333 334
Abstract Many biochemical reactions that occur within the cell are thermodynamically unfavorable. However, when these reactions are coupled to NTP (nucleoside triphosphate) hydrolysis, the energy derived from the hydrolysis of the phosphodiester bond helps drive the reaction in the favorable direction. Examples of such proteins can be found in almost all facets of cellular metabolism: glycolysis and the TCA cycle, protein Methods in Enzymology, Volume 533 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420067-8.00025-8
#
2013 Elsevier Inc. All rights reserved.
325
326
Vaishnavi Rajagopal and Jon R. Lorsch
biosynthesis, DNA and RNA metabolism, cellular trafficking, cell signaling, cell division etc. Thus, characterization of the NTPase activity of these proteins in vitro can help in understanding the role of the protein in complex cellular processes.
1. THEORY NTP hydrolysis is a simple chemical reaction involving the cleavage of the b–g phosphodiester bond to yield NDP (nucleoside diphosphate) and inorganic phosphate (Pi) (Fig. 25.1). Enzymes that catalyze this reaction are referred to as Nucleoside Triphosphate Hydrolases or simply NTPases. Although a host of different NTPases that utilize various NTPs has been identified across all domains of life, the most frequently encountered ones are the ATPases and GTPases – enzymes that hydrolyze ATP and GTP, respectively. Many radiometric, spectrophotometric, and fluorimetric assays are currently available to study the NTP hydrolysis by these enzymes. The radiometric assay involves the use of radioactive ATP or GTP, and is most widely used. These assays are easy to perform and use inexpensive and easily available reagents. They also do not require any specialized instruments and/or techniques. The assay involves carrying out the NTP hydrolysis
Figure 25.1 Schematic representation of the ATP hydrolysis reaction.
ATP and GTP Hydrolysis Assays (TLC)
327
under the desired conditions (varying enzyme concentration, varying time, varying temperature, varying substrate concentration, etc.) and then separating the hydrolyzed NDP and Pi from the unhydrolyzed NTP to analyze the extent of hydrolysis under the studied conditions. The radioactive label on the NTP can be present either on the a- or on the g-phosphate. If the label is on the a-phosphate, then the assay will monitor the generation of NDP from NTP, whereas if the label is on the g-phosphate, then the assay will monitor the generation of Pi from NTP. The separation techniques often used are chromatography and electrophoresis. In this section, we demonstrate the use of thin-layer chromatography (TLC) to separate NDP and Pi from NTP by assaying for the RNA-stimulated ATPase activity of the RNA helicase, NS3. Poly-ethyleneimine (PEI)-linked to cellulose, a weak anion exchanger, will be used as the separation matrix (stationary phase) and 0.4 M phosphate buffer, pH 3.4, will be used as the mobile phase. This assay is readily adaptable to the study of any ATPase or GTPase.
2. EQUIPMENT TLC chamber Vortex mixer Microcentrifuge Phosphorimager Micropipettors PEI ¼ Polyethyleneimine -cellulose TLC plates Phosphor screen cassette Micropipettor tips 0.65-ml microcentrifuge tubes
3. MATERIALS NS3h RNA helicase domain [g-32P]-ATP ATP-Na Formic acid (HCOOH) Potassium phosphate monobasic (KH2PO4) O-Phosphoric acid (H3PO4) Sodium hydroxide (NaOH) 3-(N-morpholino)propanesulfonic acid (MOPS) Magnesium chloride (MgCl2)
328
Vaishnavi Rajagopal and Jon R. Lorsch
Dithiothreitol (DTT) Tween-20 Poly-Uridylic acid (PolyU)
3.1. Solutions & buffers Step 1 10 Reaction buffer Component
Final concentration
Stock
Amount
MOPS-Na, pH 7.0
500 mM
2M
12.5 ml
MgCl2
50 mM
1M
2.5 ml
DTT
50 mM
1M
2.5 ml
Tween-20
1% (v/v)
10% (v/v)
5.0 ml
Add water to 50 ml
2 Reaction mix A Component
Final concentration
Stock
Amount
Reaction buffer
1
10
7.5 ml
PolyU
0.2 mg ml1
1 mg ml1
15.0 ml
NS3h
200 nM
As obtained
As required
Add water to 75 ml
2 Zero mix A Component
Final concentration
Stock
Reaction buffer
1
10
PolyU
0.2 mg ml
1
1 mg ml
Amount
2.5 ml 1
5.0 ml
Add water to 25 ml
2 Reaction mix B Component
Final concentration
Stock
Amount
Reaction buffer
1
10
7.5 ml
ATP-Na
10 mM
32
[g- P]-ATP Add water to 75 ml
0.01 mCi ml
7.5 ml
100 mM 1
10 mCi ml
1
0.8 ml
329
ATP and GTP Hydrolysis Assays (TLC)
ATPase quench solution Component
Final concentration
Stock
Amount
HCOOH
1N
4N
12.5 ml
Add water to 50 ml
Step 2 TLC running buffer Component
Final concentration
Stock
Amount
Phosphate buffer, pH 3.4
400 mM
1.0 M
400 ml
Add water to 1000 ml
4. PROTOCOL 4.1. Duration Preparation
About 2 h
Protocol
About 3 h
4.2. Preparation Purify NS3 helicase domain (NS3h) as previously described (Levin and Patel, 1999). Label 7 0.65-ml microcentrifuge tubes as 0, 10, 20, 30, 40, 50, and 60 min to indicate the time course of the experiment. Aliquot 15 ml of the ATPase quench solution into each of these tubes.
4.3. Note As indicated previously, this approach can be used for any enzyme system that involves the hydrolysis of NTP to NDP and Pi.
4.4. Caution Consult your institute Radiation Safety Officer for proper ordering, handling, and disposal of radioactive materials. See Fig. 25.2 for the flowchart of the complete protocol.
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Vaishnavi Rajagopal and Jon R. Lorsch
Figure 25.2 Flowchart of the complete protocol, including preparation.
5. STEP 1 STEADY-STATE ATP HYDROLYSIS 5.1. Overview In this step, we carry out kinetics of PolyU-stimulated ATP hydrolysis by NS3h under steady-state conditions.
5.2. Duration 90 min 1.1 Mix 50 ml Reaction mix A with 50 ml Reaction mix B and incubate the reaction at 22 C (room temperature). Start the timer for 60 min. 1.2 At the end of every 10 min, pipette out 15 ml of the reaction into the appropriately labeled microcentrifuge tube containing the ATPase quench solution. Vortex the sample, and spin down in a microcentrifuge. 1.3 Mix 15 ml of Zero mix with 15 ml of Reaction mix B and incubate the reaction at 22 C (or appropriate temperature). Start the timer for 60 min. This will yield the background rate of hydrolysis in the absence of enzyme. 1.4 At the end of 60 min, pipette out 15 ml of the reaction from Step 1.3 into the microcentrifuge tube containing the ATPase quench solution, labeled ‘0.’ Vortex the sample, and spin down in a microcentrifuge. This is the ‘minus enzyme’ control for the experiment. See Fig. 25.3 for the flowchart of Step 1.
ATP and GTP Hydrolysis Assays (TLC)
331
Figure 25.3 Flowchart of Step 1.
6. STEP 2 PREPARATION OF THE TLC PLATE AND CHAMBER 6.1. Overview In this step, we prepare the TLC plate and the chamber.
6.2. Duration 10 min 2.1 Cut the 1010 cm PEI-cellulose plate in half. Use one half for the analysis and store the other half as per the manufacturer’s recommendation. 2.2 Draw a line (using a graphite pencil), along the long edge of the plate, about 2 cm from the end. Gently mark the time-points (0–60) on the plate, each about 2 cm apart. This will serve as the baseline to spot the reactions (Fig. 25.4). 2.3 Add the TLC running buffer into the chamber such that it completely covers the bottom of the chamber and is filled to about 1 cm deep.
6.3. Tip When drawing the baseline on the TLC plate, take care not to score the PEI-cellulose layer as this will result in improper adsorbtion and poor or no separation of the samples.
6.4. Tip The TLC plate and chamber can be prepared between the reaction time-points to save preparation time.
332
Vaishnavi Rajagopal and Jon R. Lorsch
Figure 25.4 Preparation of the TLC plate.
7. STEP 3 TLC SEPARATION OF THE REACTION 7.1. Overview In this step, we carry out TLC separation of the ATPase reaction.
7.2. Duration 90 min 3.1 Spot 1 ml of each of the reaction points (0–60 min) on the corresponding points on the TLC plate. Allow to air dry completely. 3.2 Place the TLC plate in the TLC chamber and cover the chamber (Fig. 25.5). The buffer will rise along the plate through capillary action. 3.3 Once the buffer reaches to about 1 cm from the top of the plate, remove the plate from the chamber (Fig. 25.6). Allow to air dry completely. 3.4 Wrap the TLC plate in a clear plastic wrap, and expose onto a storage phosphor screen for 30–45 min. Scan the screen on a Phosphorimager and analyze the results.
7.3. Tip When placing the plate into the buffer chamber, ensure that the baseline containing the spotted samples is well above the buffer level. Otherwise, it might result in loss of the sample into the buffer.
ATP and GTP Hydrolysis Assays (TLC)
333
Figure 25.5 Running the TLC.
Figure 25.6 End of the TLC run.
7.4. Tip When spotting the samples onto the TLC plate, make sure that the pipette tip does not touch and/or damage the plate surface.
7.5. Tip When spotting the sample, spot it from as close to the plate as possible without damaging the surface. This will prevent excessive diffusion of the samples on the surface and will yield crisp spots on the chromatogram. See Fig. 25.7 for the flowchart of Step 3.
334
Vaishnavi Rajagopal and Jon R. Lorsch
Figure 25.7 Flowchart of Step 3.
REFERENCES Referenced Literature Levin, M. K., & Patel, S. S. (1999). The helicase from Hepatitis C virus is active as an oligomer. The Journal of Biological Chemistry, 274(45), 31839–31846.
ATP and GTP Hydrolysis Assays (TLC) Vaishnavi Rajagopal, Jon R. Lorsch1 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Duration 4.2 Preparation 4.3 Note 4.4 Caution 5. Step 1 Steady-state ATP Hydrolysis 5.1 Overview 5.2 Duration 6. Step 2 Preparation of the TLC Plate and Chamber 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Tip 7. Step 3 TLC Separation of the Reaction 7.1 Overview 7.2 Duration 7.3 Tip 7.4 Tip 7.5 Tip References
326 327 327 328 329 329 329 329 329 330 330 330 331 331 331 331 331 332 332 332 332 333 333 334
Abstract Many biochemical reactions that occur within the cell are thermodynamically unfavorable. However, when these reactions are coupled to NTP (nucleoside triphosphate) hydrolysis, the energy derived from the hydrolysis of the phosphodiester bond helps drive the reaction in the favorable direction. Examples of such proteins can be found in almost all facets of cellular metabolism: glycolysis and the TCA cycle, protein Methods in Enzymology, Volume 533 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420067-8.00025-8
#
2013 Elsevier Inc. All rights reserved.
325
326
Vaishnavi Rajagopal and Jon R. Lorsch
biosynthesis, DNA and RNA metabolism, cellular trafficking, cell signaling, cell division etc. Thus, characterization of the NTPase activity of these proteins in vitro can help in understanding the role of the protein in complex cellular processes.
1. THEORY NTP hydrolysis is a simple chemical reaction involving the cleavage of the b–g phosphodiester bond to yield NDP (nucleoside diphosphate) and inorganic phosphate (Pi) (Fig. 25.1). Enzymes that catalyze this reaction are referred to as Nucleoside Triphosphate Hydrolases or simply NTPases. Although a host of different NTPases that utilize various NTPs has been identified across all domains of life, the most frequently encountered ones are the ATPases and GTPases – enzymes that hydrolyze ATP and GTP, respectively. Many radiometric, spectrophotometric, and fluorimetric assays are currently available to study the NTP hydrolysis by these enzymes. The radiometric assay involves the use of radioactive ATP or GTP, and is most widely used. These assays are easy to perform and use inexpensive and easily available reagents. They also do not require any specialized instruments and/or techniques. The assay involves carrying out the NTP hydrolysis
Figure 25.1 Schematic representation of the ATP hydrolysis reaction.
ATP and GTP Hydrolysis Assays (TLC)
327
under the desired conditions (varying enzyme concentration, varying time, varying temperature, varying substrate concentration, etc.) and then separating the hydrolyzed NDP and Pi from the unhydrolyzed NTP to analyze the extent of hydrolysis under the studied conditions. The radioactive label on the NTP can be present either on the a- or on the g-phosphate. If the label is on the a-phosphate, then the assay will monitor the generation of NDP from NTP, whereas if the label is on the g-phosphate, then the assay will monitor the generation of Pi from NTP. The separation techniques often used are chromatography and electrophoresis. In this section, we demonstrate the use of thin-layer chromatography (TLC) to separate NDP and Pi from NTP by assaying for the RNA-stimulated ATPase activity of the RNA helicase, NS3. Poly-ethyleneimine (PEI)-linked to cellulose, a weak anion exchanger, will be used as the separation matrix (stationary phase) and 0.4 M phosphate buffer, pH 3.4, will be used as the mobile phase. This assay is readily adaptable to the study of any ATPase or GTPase.
2. EQUIPMENT TLC chamber Vortex mixer Microcentrifuge Phosphorimager Micropipettors PEI ¼ Polyethyleneimine -cellulose TLC plates Phosphor screen cassette Micropipettor tips 0.65-ml microcentrifuge tubes
3. MATERIALS NS3h RNA helicase domain [g-32P]-ATP ATP-Na Formic acid (HCOOH) Potassium phosphate monobasic (KH2PO4) O-Phosphoric acid (H3PO4) Sodium hydroxide (NaOH) 3-(N-morpholino)propanesulfonic acid (MOPS) Magnesium chloride (MgCl2)
328
Vaishnavi Rajagopal and Jon R. Lorsch
Dithiothreitol (DTT) Tween-20 Poly-Uridylic acid (PolyU)
3.1. Solutions & buffers Step 1 10 Reaction buffer Component
Final concentration
Stock
Amount
MOPS-Na, pH 7.0
500 mM
2M
12.5 ml
MgCl2
50 mM
1M
2.5 ml
DTT
50 mM
1M
2.5 ml
Tween-20
1% (v/v)
10% (v/v)
5.0 ml
Add water to 50 ml
2 Reaction mix A Component
Final concentration
Stock
Amount
Reaction buffer
1
10
7.5 ml
PolyU
0.2 mg ml1
1 mg ml1
15.0 ml
NS3h
200 nM
As obtained
As required
Add water to 75 ml
2 Zero mix A Component
Final concentration
Stock
Reaction buffer
1
10
PolyU
0.2 mg ml
1
1 mg ml
Amount
2.5 ml 1
5.0 ml
Add water to 25 ml
2 Reaction mix B Component
Final concentration
Stock
Amount
Reaction buffer
1
10
7.5 ml
ATP-Na
10 mM
32
[g- P]-ATP Add water to 75 ml
0.01 mCi ml
7.5 ml
100 mM 1
10 mCi ml
1
0.8 ml
329
ATP and GTP Hydrolysis Assays (TLC)
ATPase quench solution Component
Final concentration
Stock
Amount
HCOOH
1N
4N
12.5 ml
Add water to 50 ml
Step 2 TLC running buffer Component
Final concentration
Stock
Amount
Phosphate buffer, pH 3.4
400 mM
1.0 M
400 ml
Add water to 1000 ml
4. PROTOCOL 4.1. Duration Preparation
About 2 h
Protocol
About 3 h
4.2. Preparation Purify NS3 helicase domain (NS3h) as previously described (Levin and Patel, 1999). Label 7 0.65-ml microcentrifuge tubes as 0, 10, 20, 30, 40, 50, and 60 min to indicate the time course of the experiment. Aliquot 15 ml of the ATPase quench solution into each of these tubes.
4.3. Note As indicated previously, this approach can be used for any enzyme system that involves the hydrolysis of NTP to NDP and Pi.
4.4. Caution Consult your institute Radiation Safety Officer for proper ordering, handling, and disposal of radioactive materials. See Fig. 25.2 for the flowchart of the complete protocol.
330
Vaishnavi Rajagopal and Jon R. Lorsch
Figure 25.2 Flowchart of the complete protocol, including preparation.
5. STEP 1 STEADY-STATE ATP HYDROLYSIS 5.1. Overview In this step, we carry out kinetics of PolyU-stimulated ATP hydrolysis by NS3h under steady-state conditions.
5.2. Duration 90 min 1.1 Mix 50 ml Reaction mix A with 50 ml Reaction mix B and incubate the reaction at 22 C (room temperature). Start the timer for 60 min. 1.2 At the end of every 10 min, pipette out 15 ml of the reaction into the appropriately labeled microcentrifuge tube containing the ATPase quench solution. Vortex the sample, and spin down in a microcentrifuge. 1.3 Mix 15 ml of Zero mix with 15 ml of Reaction mix B and incubate the reaction at 22 C (or appropriate temperature). Start the timer for 60 min. This will yield the background rate of hydrolysis in the absence of enzyme. 1.4 At the end of 60 min, pipette out 15 ml of the reaction from Step 1.3 into the microcentrifuge tube containing the ATPase quench solution, labeled ‘0.’ Vortex the sample, and spin down in a microcentrifuge. This is the ‘minus enzyme’ control for the experiment. See Fig. 25.3 for the flowchart of Step 1.
ATP and GTP Hydrolysis Assays (TLC)
331
Figure 25.3 Flowchart of Step 1.
6. STEP 2 PREPARATION OF THE TLC PLATE AND CHAMBER 6.1. Overview In this step, we prepare the TLC plate and the chamber.
6.2. Duration 10 min 2.1 Cut the 1010 cm PEI-cellulose plate in half. Use one half for the analysis and store the other half as per the manufacturer’s recommendation. 2.2 Draw a line (using a graphite pencil), along the long edge of the plate, about 2 cm from the end. Gently mark the time-points (0–60) on the plate, each about 2 cm apart. This will serve as the baseline to spot the reactions (Fig. 25.4). 2.3 Add the TLC running buffer into the chamber such that it completely covers the bottom of the chamber and is filled to about 1 cm deep.
6.3. Tip When drawing the baseline on the TLC plate, take care not to score the PEI-cellulose layer as this will result in improper adsorbtion and poor or no separation of the samples.
6.4. Tip The TLC plate and chamber can be prepared between the reaction time-points to save preparation time.
332
Vaishnavi Rajagopal and Jon R. Lorsch
Figure 25.4 Preparation of the TLC plate.
7. STEP 3 TLC SEPARATION OF THE REACTION 7.1. Overview In this step, we carry out TLC separation of the ATPase reaction.
7.2. Duration 90 min 3.1 Spot 1 ml of each of the reaction points (0–60 min) on the corresponding points on the TLC plate. Allow to air dry completely. 3.2 Place the TLC plate in the TLC chamber and cover the chamber (Fig. 25.5). The buffer will rise along the plate through capillary action. 3.3 Once the buffer reaches to about 1 cm from the top of the plate, remove the plate from the chamber (Fig. 25.6). Allow to air dry completely. 3.4 Wrap the TLC plate in a clear plastic wrap, and expose onto a storage phosphor screen for 30–45 min. Scan the screen on a Phosphorimager and analyze the results.
7.3. Tip When placing the plate into the buffer chamber, ensure that the baseline containing the spotted samples is well above the buffer level. Otherwise, it might result in loss of the sample into the buffer.
ATP and GTP Hydrolysis Assays (TLC)
333
Figure 25.5 Running the TLC.
Figure 25.6 End of the TLC run.
7.4. Tip When spotting the samples onto the TLC plate, make sure that the pipette tip does not touch and/or damage the plate surface.
7.5. Tip When spotting the sample, spot it from as close to the plate as possible without damaging the surface. This will prevent excessive diffusion of the samples on the surface and will yield crisp spots on the chromatogram. See Fig. 25.7 for the flowchart of Step 3.
334
Vaishnavi Rajagopal and Jon R. Lorsch
Figure 25.7 Flowchart of Step 3.
REFERENCES Referenced Literature Levin, M. K., & Patel, S. S. (1999). The helicase from Hepatitis C virus is active as an oligomer. The Journal of Biological Chemistry, 274(45), 31839–31846.
