Spectroscopic and physicochemical studies on the interactions of reversible H+/K(+)-ATPase inhibitors with...

Three complementary techniques, differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, have been used to characterise the interactions between dimyristoylphosphatidylcholine (DMPC) model biological membranes and two non-covalent inhibitors of the gastric (H+, K+)-ATPase. DSC, FT-IR and deuterium NMR studies of side-chain perdeuterated DMPC (DMPC-d54) support the prediction, based on physical property measurements, that SK&F 96079 partitions readily into phospholipid bilayers, resulting in a slight but measurable disordering of the lipid hydrocarbon side-chain motion and a concomitant reduction in the co-operativity and onset temperature of the gel to liquid crystalline phase transition. However, FT-IR and deuterium NMR studies show that the bilayer structure remains intact even at high (1:4) compound to lipid molar ratios. Proton (1H) NMR nuclear Overhauser effect determinations in sonicated codispersions reveal details of the membrane bound conformations of SK&F 96079. The structurally related analogue SK&F 96464, also studied by 1H-NMR, can be shown, by interpreting the effects of nitroxide-labelled fatty acid relaxation probes, to adopt a well-defined orientation relative to the bilayer, in contrast to SK&F 96079. This orientation directs the proton at the 5-position of the quinoline ring towards the hydrophobic centre of the bilayer, and the quinoline 8-methoxy group towards the surface and hence the aqueous phase. Molecular modelling has been used to rationalise this orientation in terms of hydrogen bonds between the amino NH group of SK&F 96464 and the sn-1 carbonyl group of DMPC, and between the NH group of the protonated quinoline ring of SK&F 96464 and the DMPC phosphodiester group.