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Measuring In Vitro ATPase Activity for Enzymatic Characterization

Published: August 23rd, 2016



1Department of Microbiology and Immunology, University of Michigan

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.

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.

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 pumping1-3. 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....

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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 .......

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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.......

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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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The authors would like to acknowledge funding from a National Institutes of Health grant RO1AI049294 (to M. S.).


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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

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