Background

Adenosine triphosphatases (ATPases) are a class of enzymes that catalyze the breakdown of ATP into ADP and free inorganic phosphate (Pi). This breakdown and release of Pi generates energy used by enzymes to carry out the chemical reactions that require energy. This process is an integral part of all the kingdom of life. ABC transporters are the transmembrane proteins that move solute through the membrane by ATPase activity (Rees et al., 2009). Some ATPases are cytoplasmic or membrane-associated proteins. AAA⁺ proteins are the ATPases associated with diverse cellular activities such as protein degradation, membrane fusion, disassembly of protein complexes, microtubule dynamics, etc. (Snider et al., 2008). We identified GENERAL CONTROL NON-REPRESSIBLE-4 (GCN4), an AAA⁺-ATPase, as a novel player in regulating stomatal aperture and thus playing a role in plant innate immunity and drought tolerance (Kaundal et al., 2017). Here we describe the method to prove its ATPase activity. The GCN4 as an ATPase enzyme converts ATP to ADP and Pi, and then the enzyme phosphoribosyl phosphorylase (PNP) converts the MESG substrate into the final substrate. The assay monitors the spectrophotometric shift of the substrate (MESG) in the presence of Pi from 330 to 360 nm. The conversion rate is directly correlated with the amount of Pi present in the solution and, that depends on the conversion of ATP to ADP by GCN4 ATPase activity. We reported the ATPase activity of a recombinant protein (GCN4-His) as absorbance (ABS) at 360 nm vs. protein concentration (Kaundal et al., 2017).

Many techniques have been used to quantify the in vitro ATPase activity of purified proteins. Most of the techniques for quantification are based on the detection of free inorganic phosphate (Pi). The most common method uses the calorimeter substrate malachite green (Carter and Karl, 1982). Besides this, fluorescent and radioactive substrates are also used in various protocols to detect free phosphate in the ATPase reaction (Brune et al., 1994; Shiue et al., 2006). In this protocol, we used 2-amino-6-mercapto-7-methylpurine riboside (methylthioguanosine, a guanosine analog: MESG) as a substrate to detect the presence of free phosphate during ATPase activity (Webb, 1992). The purine nucleoside phosphorylase (PNP) enzyme in the presence of inorganic phosphate, which is released from ATP (an experimental substrate) upon hydrolysis to ADP by ATPase (experimental enzyme to be tested), converts MSEG (assay substrate) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. This enzymatic conversion of MSEG results in a spectrophotometric shift in maximum absorbance from 330 nm for the MESG substrate to 360 nm for the product 2-amino-6-mercapto-7-methylpurine (Figure 1). The advantage of this protocol is that it does not require long incubation steps and the reaction can be incubated in the plate reader itself for the required duration. The protocol is based on the ENZcheck Phosphate Assay Kit. Most of the components for enzyme assay come with the kit, including potassium phosphate standard, so the reaction is very convenient to assemble. We optimized the protocol from 1 ml reaction to 300 µl reaction to carry out in a microtiter plate. This protocol describes the detailed assay for calculating the ATPase activity of a recombinant GCN4, an AAA⁺-ATPase protein in U/ml, and specific activity. This protocol can also be used for the calculation of GTPase activity of a protein by using GTP as a substrate (Webb and Hunter, 1992).