A Characterization of the Effect of Apolipoprotein AII on the Structure and Function of Apolipoprotein AI in Reconstituted High Density Lipoproteins
Durbin, Diane Maria
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https://hdl.handle.net/2142/84882
Description
Title
A Characterization of the Effect of Apolipoprotein AII on the Structure and Function of Apolipoprotein AI in Reconstituted High Density Lipoproteins
Author(s)
Durbin, Diane Maria
Issue Date
1997
Doctoral Committee Chair(s)
Jonas, Ana
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
High density lipoproteins (HDL) can be fractionated by immunoaffinity chromatography into two main subclasses: a fraction which contains apolipoprotein (apo) AI and apoAII, and a fraction which contains apoAI but not apoAII. The effect of apoAII on the structure and function of apoAI in reconstituted HDL was investigated. ApoAII was labeled with the fluorescent probe dansyl chloride (DNS) and added to rHDL prepared with apoAI, dipalmitoyl phosphatidylcholine and cholesterol. The binding of apoAII-DNS to AIrHDL was monitored by fluorescence polarization, which showed that apoAII bound rapidly and irreversibly. The intrinsic fluorescence of apoAI enabled the detection and quantitation of displaced apoAI by the change in $\lambda$max of Trp residues in the presence of 1.5 M guanidine hydrochloride (GuHCl). The addition of apoAII to AIrHDL in a 2:1 molar ratio, apoAI:apoAII, produced complexes containing two molecules of apoAI and one molecule of apoAII, determined by chemical cross-linking. Measurement of the $\alpha$-helical content of the apoAII/AIrHDL complex by circular dichroism indicated that both apolipoproteins retained the same amount of $\alpha$-helical secondary structure as in the respective controls. Fluorescence measurements of $\lambda$max detected no change in the polarity of the Trp residues in the N-terminal half of apoAI. However, an increased susceptibility to GuHCl denaturation, and the stripping of apoAI from the hybrid rHDL under electrophoresis indicated apoAI had adopted a less stable structure in the presence of apoAII. Chemical cross-linking showed apoAI rearrangements take place to allow apoAII binding. Limited proteolytic digestion of AIrHDL with trypsin produced an N-terminal apoAI fragment of approximately 14 kDa, indicating residues in the central region of apoAI are more exposed to proteolysis after apoAII binding. The addition of apoAII to AIrHDL and LCAT resulted in a decrease in apparent $\lambda$max/Km compared to the control AIrHDL and LCAT. The data show that the binding of apoAII to AIrHDL destabilizes apoAI structure on rHDL. The presence of apoAII also affects the function of apoAI by inhibiting its activation of LCAT leading to a reduced reactivity of the apoAII/AIrHDL complex with the enzyme.
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