Differential Interaction of Cholesterol with Phosphatidylcholine on the Inner and Outer Surfaces of Lipid Bilayer Vesicles

Abstract

The Proton nuclear magnetic resonance (PMR) spectroscopy has been applied extensively in recent years to elucidate the structural or conformational properties of phospholipids and biomembranes (1). Most of these studies, however, are concerned primarily with the mobility of fatty acyl chains of the bilayer (2, 3). As reported several times (4-6), paramagnetic ions such as europium (Eug+) added to the exterior of lipid vesicles shift the field position of the -N+(CHI3)3protons of phosphatidylcholine (PC) molecules that reside on the outer surface of lipid vesicles, which are closed shelllike spheres of about 250 X in diameter, each comprised of a single, continuous, lipid-bilayer membrane enclosing a volume of aqueous solution (7). Using these paramagnetic ions as shift markers, the head groups in the two halves of vesicle bilayers can be spectrally identified. Consequently, the application of PMR technique has now been extended to investigate the structural properties of the outer and (or) inner polar head regions of the bilayer. The interaction of phospholipid with cholesterol in the bilayer structure has been studied recently by a variety of physical techniques (8-10). On the basis of these studies, it has been proposed that the effect of cholesterol on phospholipids in the bilayer structure is to restrict differentially the number of steric conformations accessible to the segments of the fatty acyl chains (11). However, more recent studies have shown that the 3,8-OH group of cholesterol is also of crucial importance for phospholipid-sterol interaction (12, 13), and, as a consequence, interactions in the polar head regions of the bilayer should be examined further.In this preliminary communication, we report the effects of cholesterol content on the -N+(CH3)3 proton resonance signal of the vesicle bilayer in the presence of a paramagnetic lanthanide ion, praseodymium ion (Pr'+). We chose Pr'+ rather than Eu8+ as a shift marker to study differentially the two polar head regions of the vesicle bilayer because, at a lower concentration, the Pr'+ ions produced at greater spectral splitting of the -N+(CH3)3 resonance signal upon coordination with the polar head groups of the bilayer. EXPERIMENTAL MATERIALS AND PROCEDURESCholesterol was obtained from Sigma Co., St. Louis, Mo., and further purified through bromination according to the method of Schwenk and Werthessen (14). L-a-Dipalmitoyl PC was purchased from Calbiochem, La Jolla, Calif. Egg PC was isolated and purified as described previously (7)

1-Anilino-8-naphthalenesulfonate: A fluorescent probe of ion and ionophore transport kinetics and trans-membrane asymmetry

“…The results of this analysis, tabulated in Table 11, show an approximate two-fold preference of cholesterol and PE for the inside surface and an approximate four-fold preference of PA-for the inside surface. The cholesterol result is in agreement with the results of Huang et al (1974) who have given evidence that egg PC bilayer membranes with overall mole fractions of cholesterol above 30 ~ have a larger mole fraction of cholesterol on the inner half of the bilayer. These workers interpreted the preference as the result of replacement of PC molecules with unsaturated chains by cholesterol on the inner layer.…”

“…Michaelson, Horwitz and Klein, (1973) have used nuclear magnetic resonance techniques to show that phosphatidyl glycerol (PG) concentrates on the inside surface of vesicles prepared from PG-PC mixtures. Huang et al (1974) have studied vesicles prepared from cholesterol-PC mixtures with a variety of physical techniques and have concluded that cholesterol distributes preferentially on the inside surface. Litman (1973) has used data on the inside-outside distribution of PE in vesicles made from PE/PC mixtures and has made calculations using estimates of the vesicle size to demonstrate a tendency for PE to be concentrated on the inside surface.…”

Morphology of egg phosphatidylcholine-cholesterol single-bilayer vesicles, studied by freeze-etch electron microscopy

“…Thus above 20-30moles cholesterol/100moles, the cholesterol composition of the outer monolayer of the vesicle remains essentially constant, with all additional cholesterol being incorporated in the inner monolayer. These conclusions are fully supported by NMR measurements of the outside-inside distribution of phospholipid molecules across the bilayer of unsaturated phosphatidylcholine vesicles containing cholesterol (Huang et al, 1974;de Kruijff, Cullis & Radda, 1975, 1976. This asymmetric packing of cholesterol means that the number of phosphatidylcholine molecules per vesicle must also increase with increasing cholesterol composition, in order not to have an unrealistically high cholesterol/phospholipid ratio in the inner monolayer.…”

“…Conversely, the asymmetric packing effect could give rise to the triggering of rapid transbilayer migration of cholesterol by changes in lateral segregation. This might be one possible mechanism by which regions of high radius of curvature could be centers of high biological activity, as suggested by Huang et al (1974).…”

“…extended bilayers or monolayers (cf. Huang et al, 1974). The area per phospholipid molecule in egg phosphatidylcholine bilayers is 62.7 A 2 in the absence of cholesterol and 46.7A 2 in the fully condensed state at 50moles cholesterol/100moles (Levine & Wilkins, 1971).…”