Characterization of Binding of Azidopyrene, a Hydrophobic, Photolabile Probe, to Escherichia Coli and Salmonella Typhimurium
Wolf, Marcia Kay
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https://hdl.handle.net/2142/71158
Description
Title
Characterization of Binding of Azidopyrene, a Hydrophobic, Photolabile Probe, to Escherichia Coli and Salmonella Typhimurium
Author(s)
Wolf, Marcia Kay
Issue Date
1982
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
The association of azidopyrene, a hydrophobic, photolabile molecule, with whole cells of Escherichia coli and Salmonella typhimurium was studied. An assay is described to quantitate irreversible light-dependent binding to cells. This binding has little or no effect on proline uptake, ATP levels, or glutamine uptake in the cells.
Conditions that result in an increased amount of azidopyrene binding include various energy poisons, EDTA/Tris treatment, and membrane mutations involving extensive changes in lipopolysaccharide. Using an E. coli uncA mutant it is shown that the amount of azidopyrene binding inversely correlates with the membrane potential but is independent of ATP levels.
Cells containing irreversibly bound azidopyrene photoproduct were fractionated. The photoproduct in E. coli and S. typhimurium deenergized and energized cells was primarily with the outer and inner membrane fractions with a varying amount with the soluble proteins. The photoproduct was not released when cells were subjected to osmotic shock. There was not a significant change in distribution in starved vs energized cells even though the amount of binding was much higher in starved cells. Little of the photoproduct comigrated with proteins in SDS polyacrylamide gels, but of the small amount that was attached to proteins, there was selective labelling of proteins. Almost all of the photoproduct extracted with cellular lipids but ran independent of cellular phospholipids in thin layer chromatography. Thus the photoproduct seems not to be covalently attached to a cell component even though it is irreversibly attached to membranes.
These data suggest a change in outer and inner membrane structure occurs in response to the membrane potential across the inner membrane which is detected by a change in association of hydrophobic molecules. In cells with extensive LPS mutations, there is some change in membrane structure such that the amount of azidopyrene binding is high even in energized cells and does not change in response to a collapsed membrane potential.
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