The ATPase assay is a membrane
assay
An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a ...
that indirectly measures the activity of
efflux transporters.
ATP Binding Cassette or efflux transporters mediate the transport of
substrates across cell membranes against a
concentration gradient
Molecular diffusion, often simply called diffusion, is the thermal motion of all (liquid or gas) particles at temperatures above absolute zero. The rate of this movement is a function of temperature, viscosity of the fluid and the size (mass) of ...
. ATP cleavage is tightly linked to substrate translocation, as the energy for the substrate translocation is derived from
ATP hydrolysis
ATP hydrolysis is the catabolic reaction process by which chemical energy that has been stored in the high-energy phosphoanhydride bonds in adenosine triphosphate (ATP) is released after splitting these bonds, for example in muscles, by prod ...
. ATP hydrolysis yields inorganic phosphate (Pi), which can be measured by a simple colorimetric reaction. The amount of Pi liberated is directly proportional to the activity of the transporter.
Use of ATPase Assay
The ATPase assay is designed to indicate the nature of the interaction between the compound and the
transporter. The ATPase assays are used in two different modes: ATPase activation and ATPase inhibition. Transported
substrates increase baseline ATPase activity, while inhibitors or slowly transported compounds inhibit baseline ATPase activity and/or the ATPase activity measured in the presence of a stimulating agent. The ATPase assays can therefore have the potential for determining whether a compound acts as a transporter substrate and/or inhibitor.
Mutations in the ATPase domain result in loss of transport function. The activity of the multidrug transporter in drug-resistant cells is associated with rapid cellular ATP depletion when ATP synthesis is inhibited.
References
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ATP-binding cassette transporters