Degree Type

Dissertation

Date of Award

1991

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Carl L. Tipton

Abstract

Two oxysterols, 20(R and S)-20,25-dihydroperoxy-5-cholesten-3[beta]-ol (HPs) isolated from the mixture of cholesterol autoxidation products have been found to reduce effectively cholesteryl ester accumulation in mouse peritoneal macrophages. The effect of HPs was associated with inhibition of cholesterol esterification by suppressing acyl-CoA:cholesterol acyltransferase activity, and by interruption of acetylated low-density-lipoprotein metabolism. The mechanism by which HPs inhibit ACAT activity is investigated in detail in the present study. With reconstituted liposomes, I demonstrated that an increase in cholesterol concentration did not prevent the inhibitory effect of HPs. On the other hand, inactivated ACAT activity was partially restored by treatment with reducing agents, including sodium borohydride, N-methyl-mercaptoacetamide, 2,3-dimercaptopropanol and dithiothreitol. These results suggest that HPs affect the enzyme activity, most likely, through oxidation rather than acting as competitive inhibitors. The reactivities of HPs were studied with model peptides and protein and it was found that the protein sulfhydryl and thioether groups were susceptible to the oxidation by HPs. With specific thiol-modification agent, phenylarsine oxide (PAO), I demonstrated the importance of vicinal sulfhydryl groups for ACAT activity. The facts that phenylarsine oxide-reacted ACAT was insensitive to further inactivation by HP-B and that the same extent of reactivation by 2,3-dimercaptopropanol could be achieved after PAO plus HP-B treatment and after PAO alone suggest that HPs and phenylarsine oxide share common reactive sites. Finally, I proposed a mechanism of two-step modification to explain how HPs inhibit ACAT activity.

DOI

https://doi.org/10.31274/rtd-180813-11677

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Meiling Shih

Language

en

Proquest ID

AAI9212188

File Format

application/pdf

File Size

106 pages

Share

COinS