Apolipoprotein A-I (apoA-I) is a major component of high-density lipoprotein (HDL), an anti-antherogenic complex responsible for reverse cholesterol transport. Previous studies have shown that lipid-free apoA-I neutralizes lipopolysaccharides (LPS), which are major constituents of the outer bacterial membrane of gram-negative bacteria, therefore reducing endotoxic effects. In addition, the protein destabilizes bilayers of negatively charged phosphatidylglycerol (PG), a phospholipid found in the inner bacterial membrane, which may result in cell lysis. Since the majority of apoA-I is found in the lipid-bound form as HDL, the binding of lipid-bound and lipid free apoA-I with LPS and PG was compared. To prepare lipid-bound apoA-I, reconstituted HDL (rHDL) was produced with apoA-I and 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Non-denaturing polyacrylamide gel electrophoreses (PAGE) showed that LPS binding was dramatically decreased for rHDL in comparison to lipid-free apoA-I. The ability to destabilize PG bilayer membranes was measured by the protein-induced release of calcein from the vesicles. This showed that while lipid-free apoA-I induced the release of 80.3% calcein, only 14.5% was released by rHDL. These results showed that when apoA-I is in the lipid-bound state, its binding with LPS and PG bilayer vesicles was decreased significantly. Thus, in order to be effective as an antimicrobial protein, apoA-I needs to be in the lipid-free conformation.
|Advisor:||Weers, Paul M. M.|
|Commitee:||Bhandari, Deepali, Schwans, Jason|
|School:||California State University, Long Beach|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||MAI 81/1(E), Masters Abstracts International|
|Keywords:||apoA-I, HDL, rHDL|
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