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Title:Isolation and Lipid Binding Properties of the Low Molecular Weight Protein Components of Bovine High Density Lipoprotein
Author(s):Patterson, Bruce Wayne
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Chemistry, Biochemistry
Abstract:Seven proteins of the low-molecular (ApoC) class of apolipoproteins have been isolated from bovine serum high density lipoprotein (HDL). Five of these (termed D(,1) through D(,5)) correspond to the five proteins reported by Lim and Scanu (Artery, 2 {1976} 483-496); the two additional proteins are termed D(,a) and D(,b). A study of the interactions between these seven apolipoproteins and dimyristoyl phosphatidylcholine (DMPC) represents the first investigation to date of the lipid binding properties of any non-human ApoC protein.
A spectroscopic examination revealed that all seven proteins underwent changes in secondary and/or tertiary structure upon the addition of lipid. This was evidenced by saturable increases or decreases in intrinsic protein fluorescence intensity during a titration of protein with DMPC unilamellar vesicles, accompanied by blue-shifts in tryptophan fluorescence wavelength maxima and increases in tryptophan fluorescence polarization. Due to a lack of sufficient samples, additional experiments were performed only with D(,2), D(,3), D(,4), and D(,a).
All four of these proteins exhibited increases in (alpha)-helicity upon the addition of DMPC, as viewed by circular dichroism spectroscopy. Each of these proteins was also able to clear DMPC multilamellar liposome turbidity from solution, which occurred with the fastest rate at temperatures equal or very close to the characteristic gel to liquid crystalline phase transition temperature of DMPC, showing the ability of bovine ApoC proteins to "solubilize" DMPC liposomes.
Gel filtration elutions of DMPC/bovine ApoC mixtures with these four proteins demonstrated that unilamellar vesicles also become disrupted. D(,2) and D(,3) were able to form a complex of essentially the same size and stoichiometry under a variety of initial lipid: protein ratios. Recombinants with D(,4) were less discrete, with the size and stoichiometry of complexes being dependent on the initial lipid: protein ratio used. The complexes formed are of a size (2 to 3 x 10('5) daltons) and weight percentage protein composition (30-40%) comparable to intact bovine HDL. Isopycnic density gradient ultracentrifugational isolations of complexes demonstrated that DMPC/bovine ApoC recombinants routinely banded within the density range of intact bovine HDL, 1.063 - 1.125 g/ml.
An analysis of lipid acyl chain fluidity and phase transition properties of isolated DMPC complexes (by monitoring the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene as a function of temperature) revealed that whereas each complex retains bilayer transition properties, the transition is broadened and its midpoint is elevated in complexes compared to pure DMPC.
Finally, an electron microscope examination of D(,3)/DMPC complexes revealed that vesicular structure has been rearranged into oblate ellipsoidal discs, which have a thickness equivalent to a single phospholipid bilayer.
This research was directed to survey the phospholipid binding properties of bovine ApoC proteins. Using techniques commonly used by other researchers in this area, lipid binding by the various bovine ApoC proteins was found to be quite similar to the lipid binding exhibited by human ApoC proteins and the ApoA-I of ApoA-II proteins of various mammalian HDL systems. This is not unexpected, of course; however, it is hoped that detailed comparisons of the lipid properties of various human and animal apolipoproteins may lead to an understanding of the structure/function relationships responsible for the physiologic roles of serum lipoproteins and each specie's transport requirements for exogenous and endogenous lipids.
Issue Date:1980
Type:Text
Language:English
Description:129 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.
URI:http://hdl.handle.net/2142/67397
Other Identifier(s):(UMI)AAI8018198
Date Available in IDEALS:2014-12-14
Date Deposited:1980


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