We propose to carry out a systematic investigation of the structural defects in lipid bilayers and their effects on the activities of reconstituted and natural membranes. The objective is to determine the role of structural heterogeneity and local lipid environment (as opposed to the bulk properties of the membrane lipids) on the properties and functions of the membrane. Using a combination of techniques (freeze fracture and in situ electron microscopy, 31P and 2H NMR, X-ray diffraction fluorescence rescence microscopy and depolarization) we plan to monitor the occurrence of domain boundaries and non-bilayer structures in reconstituted membranes and to determine the role of protein in modulating such structures. Specialized techniques (freeze fracture decoration, low dose diffraction contrast microscopy, thionphospholipid labelling for NMR and X-ray microanalysis, computer-aided distribution analysis) have been developed in our laboratories to study the spatial arrangement of these phase separated domains. Lipid mixtures which produce known molecular packing defects, including mixtures of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and cholesterol will be used in reconstitution. The ionic and glucose permeability, fusion efficiency and phospholipase susceptibility of vesicles of these lipids will be correlated with their structural defects. Ca++ATPase from rabbit sarcoplasmic reticulum, Na+K+TPase, glucose carrier protein (band 4.5) and insulin receptor from human and turkey erythrocytes, respectively, and Na+K+ATPase from rabbit kidney medulla will be reconstituted with given lipid mixtures. The incorporation efficiency, the activities of ATPase, the cytochalasin-B binding to glucose carriers and the insulin binding to receptors in reconstituted membranes will be measured to determine the roles of membrane structural defects in membrane function. The lipid composition in erythrocyte membranes and sarcoplasmic reticulum vesicles will be modified by cholesterol exchange and by phospholipid exchange proteins, so that the enhanced effects of structural defects on natural membranes may be compared to that in reconstituted systems. This study is expected to provide information that will bridge the gap between the present knowledge of structural defects in lipids and the uncertain mechanism of specific lipid effect on membranes. The results of this investigation would gain insight and provide methods to control the functions of biomembranes.
