Abstract
We isolated native high density lipoprotein (HDL) subclasses homogeneous in size and in their protein content with the objective of investigating the differences and similarities in their apolipoprotein AI (apoA-I) structures. Defined particles were isolated from ultracentrifugally prepared HDL by immunoaffinity and gel-filtration chromatography. The isolated 88-A LpAI, 106-A LpAI, 96-A LpAI/AII particles (LpAI, particles contain only apoA-I; LpAI/AII, particles contain apoA-I and apoA-II), together with a 93-A reconstituted HDL were analyzed for purity, composition, and content of apolipoprotein molecules per particle, and were examined by far and near circular dichroism and intrinsic fluorescence spectroscopic methods, as well as by reaction kinetics with lecithin:cholesterol acyltransferase. The spectroscopic analyses indicated that the secondary structures and three-dimensional arrangements of apoA-I in all these particles are remarkably similar: their tryptophan residues are located in similar nonpolar environments and become exposed to increasing concentrations of guanidine hydrochloride in comparable denaturation steps; the 60-65% alpha-helical structures in apoA-I are denatured in similar patterns with 0-5 M denaturant concentrations. However, increasing surface lipid contents and the presence of apoA-II stabilize apoA-I on the HDL particles. The reaction kinetics with lecithin:cholesterol acyltransferase are similar and slow for the isolated HDL particles, reflecting product inhibition, and/or an apoA-I conformation that is unfavorable for the activation of the lecithin:cholesterol acyltransferase reaction.
Highlights
We isolated native high density lipoprotein (HDL) subclasses homogeneous in size and in their protein content with the objective of investigating the differences and similarities itnheir apolipoprotein AI structures
Several laboratories have investigated the metabolic behavior of these two heterogeneous High density lipoproteins (HDL) subclasses (‘7-11)I.n vitro studies of the binding of LpAI and LpAI/AII to various cells, and their ability to promote cholesterol efflux from cells enriched in cholesterol, have yielded conflicting results
Barbaras et al [8]found that LpAI and LpAI/AII particles bound well to preadipocytes loaded with cholesterol, but thatonly the LpAI parsimilar: their tryptophan residues are located in simi- ticles promoted cholesterol efflux from the cells
Summary
Apolipoprotein A-I, LDL, and LCAT were prepared by routine methods [16,17,18] from human plasma donated by the Champaign County Blood Bank-Regional Health Resource Center. After removal of the NaSCN by dialysis, the particle size distribution for exposed and unexposed HDL was shown to be essentially identical by gradient gel electrophoresis (method described below). Mixtures of apoA-I to apoA-I1 in different molar ratios were run on the SDSPAGE gel along with the 96-8, LpAI/AII particle. The same samples were used to measure the intrinsic fluorescence polarization values at 25 "C with an SLM model 400 fluorescence polarization instrument using a 280-nm exciting light, 4-nm slit widths, and Corning glass 0-54 emission filters, Denaturation with solid GdnHCl was monitored by following the change in the wavelength of maximum fluorescence of the T spectra. The time required for each addition of GdnHCl, mixing, and recording of spectra was approximately 3 min This denaturation experiment was performed on two separate preparations of the 93-A rHDL and the native HDL subclasses, givingvery similar results both times. The Vmax(spp)/Km(wapaps) adjusted for any differences in enzyme concentration
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