ATPase Assay

Overview
Notes
gp44/62
gp41
References

1. Overview

The ATPase activity assay is a coupled reaction where the regeneration of hydrolyzed ATP, via phosphoenolpyruvate(PEP), Lactate dehydrogenase(LDH), and Pyruvate Kinase(PK), is coupled to the oxidation of NADH. The reaction occurs in a 1:1 ratio (1ATP hydrolyzed generates the loss of 1 NADH. Since NADH absorbs at 340nm and NAH+ does not, the result of spectrophotometer readings at OD340 over time give a negative slope.

2. Notes

NADH should be made with 0.01M NaOH
NADH is sensitive to UV-induced decomposition (store in amber tubes wrapped in foil in -80C freezer)
Use fresh ATP to minimize amount of ADP in the solution thereby reducing background activity

3. gp44/62

3.1 Conditions

Typical assay volume of 300 μL.
Buffer is 25 mM Tris-OAc, pH 7.5, 150 mM KOAc, 10 mM Mg(OAC)2, 10 mM 2-BME
Other assay components: 1 mM ATP, 0.2 mM NADH, 0.5 μM streptavidin, 3.6 U pyruvate kinase/ml assay, 2.5 U lactate dehydrogenase/ml assay, 3 mM PEP

DNA substrate is a small replication fork with biotin on the 3’ end of the template (Bio62/34/36):

5′-ACTCCTTCCGCACGTAATTTTTGACGCACGTTGT-3′
5′-ACACAGACGTACTATCATGACGCATCAGACAACGTGCGTCAAAAATTACGTGCGGAAGGAGT-BIO-3′
5′-TGGGTGGGAGGGAGTGGGATGATAGTACGTCTGTGT-3′

3.2 Assay

Continuous read on spectrophotometer at 340 nm.

Take background ATPase hydrolysis rate, 3 min.
Add 250 nM gp44/62, wait 5 minutes then take a slope.
Add 250 nM gp45 (trimer), wait 3 minutes then take a slope.
Add 250 nM Bio62/34/36 DNA Substrate.
Add 450 nM gp43, wait 3 minutes then take a slope.

3.3 Expected rates

The calculated rates using an extinction coefficient for NADH of 6220 M-1 cm-1 should be about:

+gp44/62) 5 nM ADP/sec
+gp45) 90 nM ADP/sec
+Bio62/34/36) 500 nM ADP/sec
+gp43) 125 nM ADP/sec

3.4 Calculation

Slope= change in Absorbance (OD340) /time ATPase Rate= Slope/(pathlength (1cm))*(Extinction coefficient on NADH (6.22 mM cm-1 ) ATP Hydrolysis/enzyme unit= ATPase Rate/[enzyme]

4. gp41

4.1 Conditions

Typical assay volume of 300 μL.
Buffer is 25 mM Tris-OAc, pH 7.5, 150 mM KOAc, 10 mM Mg(OAC)2, 10 mM 2-BME
Other assay components: 1 mM ATP, 0.2 mM NADH, 0.5 μM streptavidin, 3.6 U pyruvate kinase/ml assay, 2.5 U lactate dehydrogenase/ml assay, 3 mM PEP
DNA substrate is ssM13mp18 (purchased from NEB)

Here’s an example spreadsheet of how I set up the assay (when setting up, add streptavidin and diH2O volumes from the reactant tube to the substrate tube; the remainder of the reagents in the reaction tube will be added during the actual assay):

4.2 Assay

Measurements made using continuous spectrophometer absorbance at 340 nm.

Take background reading for 5 minutes.
Add 1 uM gp41 to the cuvette, mix, and incubate for 5 min at 37C. Then take slope (measurements every 30 sec for 5 min).
Add 2 nM M13mp18 ssDNA, incubate at 37C for 3 min, then take slope.
Add 2 uM gp32, incubate at 37C for 3 min, then take slope.
Add 1 uM gp59, incubate at 37C for 3 min, then take slope.

4.3 Expected rates