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....

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 sto....

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
.......

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