*5.5. ATP Hydrolysis by Purified, Reconstituted P-gp*

The rate of ATP hydrolysis by purified, reconstituted P-gp was determined spectrophotometrically by the liberation of inorganic phosphate as described [19,90]. Samples of P-gp (0.2–0.5 µg) were incubated in 96-well microplates with disodium-ATP (2 mM) and either Aβ peptide (1–50 µM) or nicardipine (10−<sup>9</sup> − 3 × 10−<sup>4</sup> M) in a total volume of 50 µL at 37 ◦C for 40 min. The absorbance (λ = 750 nm) was measured using an iMark plate reader. The activity values were normalised to the basal (i.e., substrate-free) level and plotted as a function of ligand concentration. Data in the presence of nicardipine were analysed using the general dose-response curve:

$$v = v\_{initial} + \left(v\_{final} - v\_{initial}\right) / \left(1 + 10^{\log\left(EC50 - [L]\right)}\right)$$

as described in [19], where: v is the activity, L is the compound added and EC50 is the potency of effect. Complete dose-response curves were not possible for the Aβ peptides due to poor solubility and their expense from commercial suppliers.
