Chapter 22 Nonradioactive Method for ATPase Assays

Abstract

Publisher Summary There is change in orthophosphate concentration by colorimetry. Nonradioactive ATPase assays follow the change. This happens after quenching an ATP-hydrolyzing reaction at regular time intervals and converting phosphate into a phosphomolybdate complex. A very sensitive method of this kind determines the quantity of phosphomolybdate as a complex with malachite green. The method described in this chapter, developed by Kodama and colleagues, is sensitive enough to measure 0.2–15 μM phosphate, a concentration range that other colorimetric methods cannot measure. The method is simple and reliable; the citric acid added after initiation of the coloring reaction removes excess molybdate and thereby greatly reduces the nonenzymatic hydrolysis of ATP catalyzed by molybdate. Because of its high sensitivity, this assay was used for measuring the ATPase activities of myosin and kinesin on a single microscope coverslip in in vitro motility assays. It has been used to measure the ATPase activities in Chlamydomonas inner-arm dynein fractions in which only small amounts of proteins are available. This chapter discusses the solutions and the procedure involved. There are also suggestions on precautions needed; like, because commercial sodium citrate is often contaminated with trace amounts of phosphate, it has been advised to prepare it from citric acid and NaOH, rather than purchase it. Use of tubes that have been washed with detergent and distilled water often results in a large scatter in the data, so disposable test tubes cannot be used, to be used are test tubes that have been rinsed with 0.5 N HCI and DDW. With higher concentrations of ATP, the time for the coloring reaction needs to be constant.