Date of Award
Doctor of Philosophy
Considerable attention has focused on the relationships that may exist between membrane lipids and membrane-associated enzyme activities. To explore these associations and to provide a better understanding of the role that lipids play in membrane function, a study was undertaken using Saccharomyces cerevisiae as a model system to study the lipid-protein interactions of membrane-bound enzymes. Using nutritional supplementation, the membrane level of ergosterol was increased which resulted in increased membrane tolerance against hyperthermia. Yeast mutants cho1 and opi3-3, defective in specific enzymes in the phospholipid biosynthetic pathway, were employed to study the effect of altered membrane phospholipid levels on the activities of cytochrome c oxidase, oligomycin-sensitive ATPase and plasma membrane-ATPase. The electron transfer capability of cytochrome c oxidase was found to be influenced by the altered ratio of phosphatidylcholine and phosphatidylethanolamine, indicating that a critical level of these phospholipids is required for the enzyme activity. To probe the structural specificity of phospholipids, analogues of choline and ethanolamine were used to enrich mitochondrial membranes physiologically. Kinetic analyses of the oxidase under these conditions revealed that the charge and size of phospholipid polar head groups markedly impaired electron flow from cytochrome c to the enzyme. Although DPH fluorescent polarization experiments revealed no major changes in membrane 'fluidity', possible membrane restructuring was observed when these membranes were probed with ANS. To assess the effect of fatty acids on cytochrome c oxidase, a double fatty acid auxotroph, FAI-4C, was used to manipulate the level of saturated and unsaturated fatty acids in mitochondrial membranes. Similar to phospholipid enrichment, the elevation of both saturated and unsaturated fatty acids in mitochondria also changed the low affinity kinetic phase of the oxidase. It may, therefore, follow that this enzyme site may be embedded in, or juxtaposed to the outer surface of the inner mitochondrial membrane bilayer in contrast to the high affinity site which has been shown to be significantly above the membrane plane. These results indicate that changes in membrane lipid composition can effect membrane-bound enzyme activities by changing enzyme conformation or possibly by altering subunit assembly. (Abstract shortened with permission of author.)
Fantin, Dennis John, "The Role Of Lipids In The Structure And Function Of Yeast Membranes" (1987). Digitized Theses. 1642.