Phospholipid activity and kinetics of reconstituted proteins and glycolipids in supported membranes

Artificial lipid membranes are useful models to gain insight into the processes occurring at the cell membrane, such as molecular recognition and signal transduction. These membranes with incorporated receptors also have a great potential in biosensor applications. In this study the formation of supported membranes onto planar solid supports has been investigated. The stability and activity of incorporated membrane receptors positioned in an appropriate lipid milieu has been studied. A potential use of such preparation for biosensing is discussed. The lipid films were made by the Langmuir Blodgett (LB) and the liposome fusion techniques. These supported films were characterised by ellipsometry, atomic force microscopy (AFM), surface plasmon resonance (SPR), and resonant mirro (RM) techniques. The thicknesses of the lipid films as determined by ellipsometry and AFM were in agreement with the thickness of a cell membrane. The kinetics of formation of the lipid films was studied using the optical methods SPR, RM and ellipsometry. Visualization of the supported membranes using AFM, showed that the supports were not homogenously covered with a bilayer and that these films consisted of intact or partly fused liposomes. In this investigation the proteins bacteriorhodopsin, cytochrome c oxidase, acetylcholinesterase and the nicotinic acetylcholine receptor were reconstituted into the supported membrane. The subsequent analysis showed that the proteins were individually distributed and that the activity was retained, in some cases for several weeks after immobilisation. The glycolipids, GM1, GM2, GD1b, asialo-GM1, globotriaosylceramide, lactosylceramide and galactosylceramide, were also reconstituted into supported membranes. Their specific interaction with the toxin ricin or with its B-chain was examined using SPR. The affinity of intact toxin and of its B-chain differed markedly and was pH dependent. The carbohydrate chain length and charge density of the glycolipids also influenced the affinity. The apparent kinetic constants for binding between the B-chain and the gangliosides GM1, GD1b and asialo-GM1 were estimated.