Abstract
Abstract The apoprotein separated from human serum lipoproteins of density 1.063 to 1.21 (HDL) by extraction with ethanol-ethyl ether (3:2) at -10° was shown to recombine in definite stoichiometric ratios either with the whole phospholipids of HDL or with the lecithin, phosphatidylethanolamine, and sphingomyelin fractions separated from the same lipoproteins. For the reaction, aqueous dispersions of phospholipids were obtained by sonic oscillation. HDL apoprotein maintained its binding property for phospholipids after acetylation, succinylation, reduction and alkylation, and treatment with neuraminidase, as well as in the presence of urea. The reconstituted protein-phospholipid complex acted as a substrate activator for the enzyme lipoprotein lipase obtained from chicken adipose tissue. This activity, previously reported for whole HDL, was not observed when either HDL apoprotein or phospholipids were used alone. The results are considered compatible with the concept that HDL is made of apoprotein subunits held by lipid bridges.
Highlights
Purity of each preparation was checked by both starch gel electrophoresis [3] and immunoelectrophoresis in agar gel with the use of antisera produced in the rabbit against whole human serum, low density lipoproteins of density 1.006 to 1.063, HDL, apo-HDL, albumin, or,-globulin [3, 9]
No further purification was achieved by either gel filtration (Sephadex G-25) or addition to HDL of heparin and divalent cations known to form a complex with the low density lipoproteins [10]
The present data indicate that the apoprotein of human serum high density lipoprotein, deprived of most of its lipid complement by treatment with organic solvents at low temperature, recombines spontaneously with phospholipids of the parent lipoprotein to form a complex with definite physicochemical and functional properties
Summary
Preparation and Purification of HDL-Lipoproteins of density1.063 to 1.21 were prepared and purified by ultracentrifugal flotation [3] from fresh (less than 24 hours at 4”) sera of healthy, fasted (18 hours) human Caucasian male donors, aged 22 to 30, Group A, Rh negative. Preparation and Purification of HDL-Lipoproteins of density. Purity of each preparation was checked by both starch gel electrophoresis [3] and immunoelectrophoresis in agar gel with the use of antisera produced in the rabbit against whole human serum, low density lipoproteins of density 1.006 to 1.063, HDL, apo-HDL, albumin, or -,-globulin [3, 9]. The preparations of HDL selected for the study were those which reacted with anti-HDL sera and maintained such a specificity after removal of their lipid moiety (see below). No further purification was achieved by either gel filtration (Sephadex G-25) or addition to HDL of heparin and divalent cations known to form a complex with the low density lipoproteins [10]. All HDL preparations gave similar patterns on starch gel and immunoelectrophoresis
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