A 1H NMR study of the effects of metal ions, cholesterol and [formula omitted] on phase transitions in the inner and outer monolayers of phospholipid vesicular membranes

Abstract

A novel method is described which allows study of the effect of various asymmetries across a phospholipid bilayer. High resolution, 1H NMR spectra of dipalmitoyl phosphatidylcholine single-bilayer vesicles are obtained at various temperatures in the presence of the lanthanide ions Pr 3+ and Gd 3+. The spectra are used to detect separately the phase transitions which occur in each monolayer of the bilayer. 1. 1. The separate 1H NMR signals from the inner and outer head-groups of the vesicles are obtained using a concentration of 6 mM Pr 3+ in the extravesicular solution. The effect of addition of other ions, Ca 2+, Mg 2+ and K +, to the extra- or intravesicular solution is studied. It is found that interaction of metal ions with the head-groups of one monolayer can be transmitted to the other monolayer and alters the temperature and extent of the phase transitions and pre-transitions observed in each layer. If a suitable metal ion concentration gradient is set up across the bilayer then the two monolayers can be in different phases at temperatures near the phase transition. 2. 2. Using 0.2 mM Gd 3+ in the extra- or intravesicular solutions indicates that there are differences between the phase behaviour and mobility of the lipid in the inner and outer monolayers, which can be attributed to their difference in curvature. 3. 3. Dipalmitoyl phosphatidylcholine vesicles containing 0–50 mol% cholesterol are studied using the same techniques and the effect of increasing concentration of cholesterol on each monolayer is observed. The data obtained indicate that even for a symmetrical distribution of cholesterol its presence can reverse the effect of a metal ion concentration gradient on the relative fluidity of the two monolayers. At 15 mol% cholesterol the bilayer is very susceptible to lysis at temperatures near the phase transition. 4. 4. When n- alkanes are incorporated into dipalmitoyl phosphatidylcholine bilayers they effect the temperature of the phase transitions, depending on their chain length. The stability of the bilayer is also affected, but only at temperatures near T c , and by alkanes of similar hydrocarbon chain length to those in the acyl chains of the lipid. There is thus a striking correlation between the chain length-dependent effect of alkanes as promotors of chemical carcinogenesis and their ability to de-stabilise the phospholipid bilayer.