Degree Name

Master of Science (MS)

Semester of Degree Completion

1977

Thesis Director

Eugene B. Krehbiel

Abstract

The ciliated protozoan Tetrahymena pyriformis-Strain W desaturated 14C-labeled fatty acid substrate in both sterol supplemented (ergosterol) and nonsupplemented cultures. Cis-octadecenoic acid (14C-9-18:1), when added to the medium during the log phase of growth, was incorporated into the cells and also desaturated at the Δ6 and Δ12 positions. The 14C labeled substrate and products, linoleate (9,12-18:2) or linolenate (6,9,12-18:3) were recovered and separated by silver-nitrate Unisil column chromatography. Initially, recovery of 14C compounds varied widely when expressed as the percent of total counts per minute recovered from column chromotography, and a variety of procedural modifications were required before a reproducibly significant effect of the foreign sterol on desaturation of substrate was demonstrated.

Addition of ergosterol to the growth medium of Tetrahymena leads to incorporation of the foreign sterol within cell membranes and supression of synthesis of the native compound tetrahymanol, as well as to changes in the fatty acid compositions of several major classes of membrane lipid. Alteration of fatty acid composition is thought to represent a regulatory mechanism whereby optimum membrane fluidity is maintained when the slightly dissimilar foreign sterol is incorporated into the phospholipid bilayer of the membranes.

The present study, using several different conditions of growth temperature, culture size and ergosterol concentrations, is an attempt to provide evidence supporting a regulatory role for membrane-bound sterol. The regulatory mechanism hypothesized is allosteric modification of an enzyme or enzymes involved in the synthesis of fatty acids contained in membrane phospholipids by the membrane-bound sterol. Such a mechanism could account, at least in part, for the balance between sterol and fatty acid content of membranes. The data presented here show that a statistically significant decrease in desaturation of 14C-labeled 9-18:1 can be demonstrated in Tetrahymena cell cultures whose membranes contain the foreign sterol ergosterol when compared to cell cultures not receiving sterol supplementation when growth temperature is maintained at 20°.

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