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|Title:||The Effect of Phospholipid Polar Head Group Supplementation on The Microsomal Cytochrome B5 System|
|Author(s):||Gilmore, James Reid|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The maintenance of a fluid phospholipid bilayer is important for a number of membrane associated functions. The fluidity of the bilayer is determined by the lipid composition. The lipid composition can be described in terms of the phospholipid polar head group composition, the fatty acid composition and the sterol content of the membrane. This study concerns the influence of the phospholipid polar head group composition upon the fluidity of LM cell membranes.
The phospholipid polar head group composition of mouse LM cells was modified by supplementation of the growth medium with ethanolamine in place of choline. Ethanolamine supplementation resulted in the incorporation of the corresponding phospholipid, phosphatidylethanolamine, into the membranes of LM cells. The fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to determine the effect of ethanolamine supplementation upon membrane fluidity. The rotational relaxation time of DPH was determined in plasma membranes, endoplasmic reticulum membranes, and mitochondrial membranes from cells supplemented with choline or ethanolamine. Ethanolamine supplementation resulted in an increase in the rotational relaxation time of DPH in the isolated membrane fractions. An increase in the rotational relaxation time of DPH in the membranes is indicative of an increase in membrane viscosity. The examination of the rotational relaxation time of DPH in whole lipid and phospholipid dispersions indicated that the increase in membrane viscosity was due to changes in the phospholipid polar head group composition of the membrane.
To determine the effect of changes in membrane fluidity upon membrane function, the microsomal cytochrome b(,5) system was selected. This electron transport system consists of cytochrome b(,5) and cytochrome b(,5) reductase. The NADH-dependent cytochrome c reductase activity is a measure of the interaction of cytochrome b(,5) and cytochrome b(,5) reductase. Ethanolamine supplemented endoplasmic reticulum membranes had a 1.8-fold lower NADH-dependent cytochrome c reductase activity. This decrease in activity was due to both a decrease in the membrane content of cytochrome b(,5) reductase and an increase in membrane viscosity.
A fluorescent cytochrome b(,5) derivative was prepared using 5-dimethylamino-1-naphthalene sulfonyl chloride. The rotational relaxation time of the labeled protein was determined in aqueous buffer, in sodium deoxycholate micelles, and when bound to to synthetic phosphatidylcholine vesicles. The protein showed essentially no rotational motion in phospholipid vesicles below the gel to liquid crystalline phase transition temperature of the phospholipid. At temperatures above the phase transition of the phospholipid the motion of the protein was much more rapid. The rotational relaxation time of the labeled protein was determined when bound to phospholipid vesicles prepared from endoplasmic reticulum membranes isolated from choline and ethanolamine supplemented cells. The longer rotational relaxation times observed in the phospholipids isolated from the ethanolamine supplemented cells was consistent with the observations made previously using DPH as a probe.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.
|Date Available in IDEALS:||2014-12-14|