The isolation and characterization of high-density-lipoprotein subfractions containing apolipoprotein E from human plasma

Abstract

1. Plasma high-density lipoprotein (HDL) was separated by heparin-Sepharose affinity chromatography into a non-bound, apolipoprotein E-poor, and a bound, apolipoprotein E-rich, fraction through the binding effect of Mn2+ in the column buffer. 2. The application of a series of elution buffers in which the concentration of Mn2+ was progressively replaced by Mg2+ resulted in the separation of the bound HDL into five subfractions. 3. Each subfraction migrated a different distance on gradient-gel electrophoresis. Three of the subfractions had RF (relative migration compared with BSA) values within the range of HDL2b. One subfraction contained largely HDL2a, with some material in the regions of HDL2b and HDL3a, and one subfraction spanned the RF regions of HDL2a, HDL3a and HDL3b. 4. The number of molecules, per HDL particle, of cholesteryl ester, non-esterified cholesterol and phospholipid increased with particle size, whereas triacylglycerol passed through a maximum and the number of amino acid residues remained approximately the same. 5. Apolipoprotein (apo) A-I was the major apoprotein in all five subfractions, but the latter differed appreciably in their contents of apo A-II and apo E. 6. The major fatty acid component of each subfraction was linoleic acid, with moderate amounts of C16:0 and C18:1 fatty acids and a smaller content of C18:0, C20:4,n-6 and C22:6,n-3, with no significant difference in composition between the subfractions. 7. This paper provides the first description of a method for the isolation of three subfractions of HDL2b together with other subfractions in quantities that are sufficient for further analytical or metabolic studies.

Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver

“…Compared with HDL, LDL are particularly enriched in steryl molecules, which makes these lipoproteins a beneficial source of cholesterol for these parasites. Among the lipoproteins used in this study, human LDL contain ∼350 and ∼1400 molecules of free and esterified cholesterol per particle, respectively, whereas human HDL contain only ∼30 and ∼70 molecules of free and esterified cholesterol per particle respectively (Wilson et al ., 1992). In contrast to intrahepatic Plasmodium , Toxoplasma cannot divert cholesterol synthesized de novo by the host cell.…”

Apolipoprotein L, a New Human High Density Lipoprotein Apolipoprotein Expressed by the Pancreas

“…9). However, it is interesting to note the discordance of the data between HDL purified by ultracentrifugation and the lipoprotein purified by immunoaffinity, showing the protein redistribution occurring during ultracentrifugation (13)(14)(15)(33)(34)(35). We found apoL to be preponderantly in HDL 3 ; however, the Lp(L) particles isolated by selected immunosorption exhibited heterogeneity of size.…”

“…Since first proposed by Alaupovic (16) protein constituents that dissociate during isolation by ultracentrifugation (13)(14)(15)(33)(34)(35). In this study, we combined liquid-phase isoelectric focusing and high resolution two-dimensional gel electrophoresis to surmount the problem posed by the predominance of apoA-I in the immunoisolated Lp(A-I) fractions that would otherwise hinder purification of proteins present at lower concentrations.…”

“…VLDL, IDL, LDL, HDL 2 , and HDL 3 were isolated by conventional sequential ultracentrifugation. Because this technique is known to disrupt lipoprotein structure (13)(14)(15)(33)(34)(35), we attempted to minimize lipoprotein alteration by submitting each lipoprotein class to equivalent ultracentrifugal forces as the 2 t product, using the same rotor. Qualitative detection of apoL in the different subclasses was maximized by immunoblotting samples overloaded on SDSpolyacrylamide gel (100 g of each fraction).…”

“…The numerous HDL molecular species are not fully apparent when HDL is prepared by ultracentrifugation. Hydrostatic pressure developed in the ultracentrifuge causes the dissociation of a portion of the complement of apolipoproteins (such as apolipoproteins A-I, A-II, C, and E) from HDL and leads to concomitant protein and lipid rearrangements (13)(14)(15)(33)(34)(35). The contents of proteins such as lecithin:cholesterol acyltransferase and cholesterol ester transfer protein shown to interact and to form physical complexes with apoA-I-containing lipoproteins are diminished or totally depleted in HDL altered by ultracentrifugal isolation (15,18).…”