What are ATPases?
Many people are familiar with the abbreviation ATP, which stands for the molecule adenosine triphosphate. ATP is the fascinating energy carrier of cellular processes and is involved in energy transfer in all living organisms. The energy released upon the chemical transformation from ATP to ADP is used in a myriad of biochemical processes. A general diagram of the conversion of ATP to ADP is depicted here.
Less well known perhaps is the enzyme that catalyzes the reaction from ATP to ADP, called an ATPase, a large macromolecular biological catalyst. The reaction proceeds slowly if at all in the absence of enzyme, but proceeds well in the presence of the ATPase. Enzymes can be thought of as biological catalysts, as they lower the activation energy required for the reaction to proceed.
Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a principal role in a wide variety of cellular functions. They are very large macromolecules like the ATPase depicted here:
ATPase F1 complex gamma subunit, which forms the central shaft that connects the F0 rotary motor to the F1 catalytic core.
In the color figure above the ribbons indicate alpha helices and the arrows are beta turns, folds, and sheets. The helices, turns, folds, and sheets are structural characteristics of the polypeptide protein chain making up the biological macromolecule. The different colors are used to indicate different chains of the macromolecule for easier viewing.
ATPases Have a Wide Variety of Functions
There are different kinds of ATPases, reflecting a variety of life-sustaining reactions such as:
- Active transport by pumping ions or phospholipids across a membrane
- Synthesis of biological macromolecules, such as DNA, RNA, proteins, and lipids
- Mechanical work, like muscle contraction, rearrangement of the cytoskeleton, and motion of cilia.
- Protein folding and chaperoning
- Protein homeostasis
The different kinds of ATPases can not only differ in function, but also in structure and the type of ion it transports. ATPases are also druggable, meaning that they are molecular targets for drug discovery and development. The most well-known ATPase inhibitor is omeprazole, a popular pharmaceutical that targets the H+/K+-ATPase system by its selective and irreversible inhibition of the proton pump in the gastric parietal cells. Other ATPase therapeutics are on the market or in development.
ATPase Activity Assays
One of the most robust uses of measuring ATPase inhibition is with an ATPase Activity Assay. The protocol developed by BellBrook Labs has been in use for over ten years, with millions of tests performed. All with the goal of first-in-class drugs targeting cancer, autoimmunity pathways, and many other indications.
The Transcreener ATPase activity assay allows the direct determination of ADP produced by enzymatic ATP hydrolysis. Simply put, the assay works by measuring the ADP produced in the reaction from ATP. The amount of ADP released is an indicator of robustness of the reaction, the more ADP detected, the more active the enzyme. The assay can allow investigators to determine if the molecule being probed is a good candidate that should be pursued as a new clinically relevant drug. The assay protocol is carried out by the Transcreener ADP assay kit is sold as a stand-alone product for investigators, so they can focus on new discovery and not assay development.
Advancing Drug Discovery and Target Validation
Prior to screening and drug discovery, a target must be ‘validated’, that is, shown to be a reasonable drug target that by modulating in some way will have a therapeutic effect in humans. The assay can be used to study ATPase activity to better understand each enzyme’s impact on biological systems.
The assay can be used for a variety of applications to create data which can be examined for certain characteristics that typically lead to a favorable drug candidate.
Applications of the ATPase activity assay include the ability to:
- Screen chemical compound libraries for enzyme inhibitors
- Determine inhibitor selectivity between enzymes
- Measure inhibitor potency for SAR
- Determine residence times for inhibitors
Advantages of the ATPase Activity Assay
The Transcreener fluorescent assay protocol reduces interference and false positives. It also eliminates the need for radioactive components, diminishing safety and disposal concerns. In addition, the ATPase assay allows sensitive detection over a broad ATP range. Both the reagents used and the signal detected are stable, allowing the flexibility to perform large automated screens. These advantages indicate that the ATPase activity assay is a great fit for high throughput screening and drug discovery.