An automated method for measuring adenosine triphosphatase (ATPase) activity is described. A modified version of a Technicon Autoanalyzer utilizing a sensitive colourimetric technique for determining inorganic phosphate concentrations (1 nmol/ml) allows investigations on enzymes of low specific activities. Dialysis may be used for measuring tissue homogenate activities or bypassed by examining purified enzyme preparations. When combined with a gradient apparatus, the proposed technique is particularly well suited for the study of enzyme kinetics in relation to cation or anion concentrations.
In recent years there have been numerous investigations on the relationship between ionic transport across various cellular membranes and the presence and characteristics of adenosine triphosphatases (ATPases), mainly the (Na+ + K+)-dependent ATPase but also the Ca2+-, K+-, or anion-dependent enzymes. In our laboratory, studies on the fish gill ATPases carried out over several years (1) have necessitated the development of an automated assay with a modified Technicon Autoanalyzer.
This fully automated method is suitable not only for the routine determination of enzyme specific activity but also for kinetic studies on any type of ATPase. Kline et al. (2) and more recently Josephson et al. (31, Stewart (4), and Dale et al. (5) have described automated assays for (Na+ + K+)ATPase. Only the technique developed by Josephson et al.
(3) permits kinetic analyses to be carried out in a simple manner. In this method, however, the protein precipitate which appears when the reaction is stopped is not removed and thus could interfere with the absorbance measurement.
The method described in this paper does not need numerous and complicated reagents and is based on: a modification of a Technicon sampler, which allows for absolute synchronization in the sampling of three different reaction components: enzyme, substrate, and ions or r Maitre-Assistant.