Measuring enzyme activities in crude homogenates: Na⁺/K⁺-ATPase as a case study in optimizing assays

In this review of assays of Na⁺/K⁺-ATPase (NKA), we explore the choices made by researchers assaying the enzyme to investigate its role in physiological regulation. We survey NKA structure and function in the context of how it is typically assayed, and how technical choices influence what can be said about the enzyme. In comparing different methods for extraction and assay of NKA, we identified a series of common pitfalls that compromise the veracity of results. We include experimental work to directly demonstrate how choices in detergents, salts and substrates influence NKA activities measured in crude homogenates. Our review of assay approaches integrates what is known from enzymology, biomedical physiology, cell biology and evolutionary biology, offering a more robust method for assaying the enzyme in meaningful ways, identifying caveats and future directions to explore its structure and function. The goal is to provide the sort of background on the enzyme that should be considered in exploring the function of the enzyme in comparative physiology.