Hydrocarbon phase transitions and lipid-protein interactions in the erythrocyte membrane. A 31P NMR and fluorescence study

Abstract

1. 1. 31P NMR and other techniques have been employed to study various derivatives of the human erythrocyte ghost membrane, derived liposomes and liposomes composed of mixtures of synthetic phosphatidylcholines. It is shown that the previously reported (Cullis, P.R. (1976) FEBS Lett. 68, 173–176) phospholipid phase transition observed in ether extracted ghosts is also observable employing fluorescence depolarization techniques. This phase transition can be (reversibly) removed by re-addition of cholesterol. 2. 2. A new semi-biological model membrane system is described which may be obtained by proteolytic digestion of peripheral membrane protein and a subsequent ether extraction. Freeze-etch studies and other considerations indicate that these “pronase digested ether extracted ghosts” consist of segments of integral membrane protein surrounded by one or two boundary layers of lipid. 3. 3. Within the terms of the “fluid mosaic” model of biological membranes lipids may exist in at least four different environments. These include unperturbed bilayer regions, bilayer regions shielded by peripheral membrane protein, lipid experiencing strong polar interactions with membrane protein (bound lipid), and lipid associated with integral membrane protein (boundary lipid). The results obtained here are consistent with the following general conclusions. 3.1. (a). The ether extraction procedure removes most, if not all, of the unperturbed bilayer lipid. 3.2. (b). The amount of membrane phospholipid “bound” to membrane protein so as to cause immobilization in the phosphate group region (on the time scale of 10 −5 s) is at most 3% of the membrane phospholipid. 3.3. (c). Phospholipids in all three of the major classes of lipid (unperturbed bilayer, shielded bilayer and boundary lipid) exhibit 31P NMR spectra consistent with bilayer structure. 3.4. (d). The motion (on the time scale of 10 −5 s) in the phosphate group region of membrane phospholipids is not sensitive to the presence of peripheral membrane protein. 3.5. (e). The phase transition behaviour and 31P NMR lineshapes observed suggest that in the ether extracted ghost system phospholipids in bilayer regions (possibly consisting largely of sphingomyelin) enter the gel state below 20°C. Below 20°C “boundary” phospholipids also experience restricted motion. Above 20°C the results would be consistent with a model whereby boundary phospholipids experience fast exchange with lipids in liquid crystalline bilayer regions.