Ouabain solution preparation

Figure 32. Time course of generation of 5 -ATP-induced membrane potential and disappearance of the membrane potential by adding a specific inhibitor ouabain for a single preparation of Na, K -ATPase-based BLM. 5 -ATP was injected into the cis side solution (final concentration 10 mM) and ouabain (DMSO solution) into the trans side solution (final concentration 1 mM) (reprinted with permission from Anal. Chim. Acta 1993, 281, 583. Copyright 1993 Elsevier Science Ltd.).

Membrane fragments containing the Na-K ATPase were obtained from frozen rabbit kidneys by a modification of the method of Kinsolving et al. (8). Mg2+ and ethylenediaminetetraacetic acid (EDTA) were omitted from all wash solutions, and the enzyme was stored in 0.02M tris-acetate buffer of pH 6.8. A second preparation was obtained from calf brain by the method of Skou and Hilberg (9). Assays were conducted by adding the enzyme preparation to solutions containing Na+, K heavy metal ions, and ATP. The difference in the rate of release of phosphate ions, estimated by the method of Taussky and Shorr (10), in the absence and the presence of ouabain was taken as a measure of Na-K ATPase activity. 

The activities of Na -K", Mg and Ca ATPases were estimated in the organ by using the method of Watson and Beamish (1981). A 1% tissue homogenate was prepared in 0.25 M ice-cold sucrose solution and centrifuged for 10 min. The super-natent was taken and three sets of incubation mixtures were prepared. In a total volume of 2 ml for all the three sets, 100 mM disodium ATP, 20 mM KCl, 100 mM NaCI, 3 mM MgCL, and 0.3 ml enzyme extract were added. Three millimolar MgCL (for Na -K ATPase) to the first set, 1 Mm ouabain (for Mg ATPase) to the... 

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