Background

Through all the kingdoms of life, ATPases are the common enzyme class to catalyze reactions that would otherwise not take place due to high energy barriers. All ATPases use the dephosphorylation of ATP to ADP, resulting in the release of inorganic phosphate. Additionally, the decomposition of ATP provides energy that ATPases use for conformational changes in a variety of ways. Since most ATPases are transmembrane proteins, conformational changes enable the transport of substrates along the membrane and even against the concentration gradient.

Despite intensive research, many details on the mechanism(s) of these ubiquitous enzymes remain to be elucidated. The cellular environment is too complex to investigate one specific protein without the influence of other proteins or molecules. Therefore, most research uses a purification approach to investigate the protein of interest exclusively in the (detergent)-solubilized state or after reconstitution in model membranes, in the case of transmembrane proteins. Since substrate transport is coupled to ATP hydrolysis, one major aspect of the protein characterization is the ATPase activity. Furthermore, it is stimulated by the substrate, pH, ionic strength of the buffer, or in the case of transmembrane proteins, the lipid surroundings.

Many techniques have been used to quantify the in vitro ATPase activity of purified proteins. The most common approach to determine the ATP hydrolysis activity of ATPases is the Malachite green assay, developed in 1966 by Itaya and Ui (Itaya and Ui, 1966). This assay has been improved several times over the last few decades by different groups (Carter and Karl, 1982; Baykov et al., 1988; Geladopoulos et al., 1991; Feng et al., 2011). The method relies on the formation of a green complex when malachite green/molybdate reacts with inorganic phosphate under acidic conditions. The amount of green molybdophosphoric acid complexes can be measured with a spectrophotometer and is directly correlated with the concentration of free inorganic phosphate in the reaction. Another widely used ATP hydrolysis assay is the NADH-coupled assay (Warren et al., 1974). Here, ATP is regenerated by the pyruvate kinase, which simultaneously converts phosphoenolpyruvate to pyruvate. The latter is subsequently converted to lactate by the lactate dehydrogenase and coupled to the oxidation of NADH to NAD⁺. The absorption spectra of both substances at 340 nm vary significantly, allowing live absorbance- or fluorescence-based tracking of NADH consumption, and therefore, indirectly, the coupled ATP hydrolysis (Radnai et al., 2019). Based on the same principle, other fluorescence-based assays use a coupled enzyme system that catalyzes a reaction of a fluorescent substrate with a free phosphate to a product with low fluorescence (Banik and Roy, 1990). Other assays are based on a fluorescent substrate to detect free phosphate in the ATPase reaction that loses its fluorescence by binding to phosphate (Brune et al., 1994). In general, assays utilizing coupled enzyme systems have the disadvantage of being sensitive to the assay conditions, e.g., pH and/or presence of lipids. Furthermore, some ATPases cannot be purified in reasonable quantities, or their ATPase activity is low. Thus, large amounts of purified protein would be needed in the experiment, since neither the Malachite green, the NADH-coupled assay, nor the fluorescence-based assays are sensitive enough with detection limits in the low micromolar or maximum nanomolar range.

Here, we describe an in vitro ATPase assay utilizing radiolabeled [γ-³²P]-ATP based on previous protocols (Gorbulev et al., 2001), with the ability to detect free phosphate in the femtomolar range. In the assay presented, the active ATPase will liberate radiolabeled gamma-phosphate from [γ-³²P]-ATP. Excess radiolabeled ATP is subsequently separated from liberated radiolabeled gamma-phosphate by molybdate-phosphate extraction, in which the molybdate-phosphate complex and non-hydrolyzed ATP partition into the organic and aqueous phase, respectively. The radioactive assay provides a direct and sensitive quantification of ATPase activity. In contrast to colorimetric and fluorescent assays, this assay is not disturbed by turbidity, which can be caused, e.g., by detergents and/or lipids present in the samples. The major limitations of radioactive ATPase assays are the hazards of handling radiolabeled isotopes and the unsuitability of this assay format for large-scale high-throughput screening.