Incorporation of cholesterol into serum high and low density lipoproteins

Excess cholesterol (CHL) was incorporated into serum lipoproteins by incubation with Celite^®-dispersed CHL or CHL crystals. The initial rates of uptake were 15 and 7 mol CHL/mol lipoprotein × h^? for low (LDL) and high (HDL) density lipoprotein, respectively. Saturation values were obtained after 48 h and were 90 and 65 mol CHL/mol LDL, and 42 and 32 mol/mol HDL using CHL on Celite and CHL cyrstals, respectively. Characterization of the lipo-proteins showed a small change in electrophoretic mobility and an increase in molecular masses, especially after incubation with CHL on Celite. Spectroscopic methods showed only minor effects on the protein moieties.     

Flotation equilibrium of serum low density lipoproteins

The molecular weight of human serum low density lipoprotein (LDL) was determined for the first time by sedimentation equilibrium or, more accurately, flotation equilibrium, in high concentration of KBrNaBr containing Tris-Cl buffer plus EDTA (density = 1.20 – 1.49 g/ml). Assuming both the molecular weight and the partial specific volume were unknown, the results at different densities gave a value of 2.87 ± 0.12 × 10⁶ for the molecular weight and 0.965 ± 0.014 ml/g for the partial specific volume.     

Studies on pig serum lipoproteins. V. Optical properties of low density lipoproteins.

The circular dichroism (CD), optical rotatory dispersion (ORD), and fluorescence emission spectra of two subfractions of pig serum low density lipoproteins (LDL1 and LDL2) were compared. The contribution of the carbohydrate moiety to the CD and ORD spectra was estimated on the basis of data obtained from isolated glycopeptides and the constituent monosaccharides. The carbohydrate moiety had no effect on the conformation of the protein moieties of LDL1 and LDL2 (apoLDL1 and apoLDL2). However, the intensities of the observed extrema in the CD and ORD spectra of the glycopeptides were greater than those expected from the monosaccharide composition. This suggests the existence of secondary structure in the carbohydrate moiety. In contrast to the carbohydrate moiety, the contribution of the lipid moiety to the CD and ORD spectra could not be neglected. When the effect of the lipid moiety was subtrated from the CD and ORD spectra, the spectra due to apoLDL1 and apoLDL2 were quite similar. Delipidation in the presence of sodium dodecyl sulfate (SDS) induced an increase in the content of disordered structure and alpha-helix accompanied by a decrease in the beta-structure. In the presence of SDS, marked quenching occurred in the fluorescence emission spectra with a blue shift of the maximum emission wavelength from 332 to 326 nm. ApoLDL1 and apoLDL2 showed quite similar SDS-induced conformational transitions. The secondary structures of apoLDL1 and apoLDL2 in the native lipoproteins were stable to changes of pH and temperature. However, this stability was lost in the presence of SDS. These results suggest the importance of the lipid moiety in maintaining the native secondary structures of LDL1 and LDL2. From the overall similarity of the optical properties of apoLDL1 and apoLDL2, we conclude that the secondary structures of apoLDL1 and apoLDL2 are identical.     

An alternative procedure for incorporating radiolabelled cholesteryl ester into human plasma lipoproteins in vitro.

A simple method has been developed for labelling human plasma lipoproteins to high specific radioactivity with radioactive cholesteryl esters in vitro. After isolation by preparative ultracentrifugation, the selected lipoprotein was incubated for 30 min at 4 degrees C in human serum (d greater than 1.215) that had been prelabelled with [4-14C]cholesteryl oleate or [1,2-3H]cholesteryl linoleate, and was then re-isolated by ultracentrifugation. All major lipoprotein classes were labelled by the procedure. Specific radioactivities of up to 18 d.p.m. . pmol-1 (46 d.p.m. . ng-1) were achieved. When radiolabelled high-density lipoprotein was infused intravenously, the radioactive cholesteryl ester behaved in vivo indistinguishably from endogenous cholesteryl esters produced by the lecithin (phosphatidylcholine): cholesterol acyltransferase reaction.     

Secondary structure in very low density and intermediate density lipoproteins of human serum.

We have studies the secondary structures of the protein moieties of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) of human serum by circular dichroism (CD). Two potential complications in the application of this technique to lipoproteins have been evaluated. First, using chronographic potentiometry in CD measurements of VLDL fractions of different mean particle diameters, we have analyzed statistically the CD signals in order to define the limits imposed by light scattering with respect to both particle diameter and wavelength. We found that CD measurements can be made to as low as 210 nm on particles of 520 A or smaller, and to 194 nm on particles of 450 A and below. Second, we have evaluated the CD contribution of lipid chromophores. Despite the high ratio of lipid to protein, the relative CD effect of the lipids is smaller than for low density lipoproteins (LDL). due to the extremely small ellipticity of natural VLDL triglycerides. Thus, CD measurements can be obtained with confidence on the preponderant bulk of normal VLDL. For the first time we report the CD spectra of human VLDL and IDL. In contrast with human LDL and the lipoproteins of the hypercholesterolemic rabbit, the entire CD SPECTRUM OF HUMAN VLDL shows increased ellipticity with decreasing temperature, which is completely reversible. We have found that the protein moieties of human VLDL and IDL contain substantially more helix (approximately 50%) than does that of human LDL.     

In vitro incorporation of radiolabeled cholesteryl esters into high and low density lipoproteins

We have developed and validated a method for in vitro incorporation of radiolabeled cholesteryl esters into low density (LDL) and high density lipoproteins (HDL). Radiolabeled cholesteryl esters dissolved in absolute ethanol were mixed with LDL or HDL in the presence of lipoprotein-deficient serum (LPDS) as a source of core lipid transfer activity. The efficiency of incorporation was dependent on: a) the core lipid transfer activity and quantity of LPDS, b) the mass of added radiolabeled cholesteryl esters, c) the length of incubation, and d) the amount of acceptor lipoprotein cholesterol. The tracer incorporation was documented by repeat density gradient ultracentrifugation, agarose gel electrophoresis, and precipitation with heparin-MnCl2. The radiolabeling conditions did not affect the following properties of the lipoproteins: 1) chemical composition, 2) electrophoretic mobility on agarose gels, 3) hydrated density, 4) distribution of apoproteins on SDS gels, 5) plasma clearance rates, and 6) immunoprecipitability of HDL apoproteins A-I and A-II. Rat HDL containing radiolabeled cholesteryl esters incorporated in vitro had plasma disappearance rates identical to HDL radiolabeled in vivo.     

Hepatic triglyceride lipase promotes low density lipoprotein receptor-mediated catabolism of very low density lipoproteins in vitro.

We demonstrate here that hepatic triglyceride lipase (HTGL) enhances VLDL degradation in cultured cells by a LDL receptor-mediated mechanism. VLDL binding at 4 degrees C and degradation at 37 degrees C by normal fibroblasts was stimulated by HTGL in a dose-dependent manner. A maximum increase of up to 7-fold was seen at 10 microg/ml HTGL. Both VLDL binding and degradation were significantly increased (4-fold) when LDL receptors were up-regulated by treatment with lovastatin. HTGL also stimulated VLDL degradation by LDL receptor-deficient FH fibroblasts but the level of maximal degradation was 40-fold lower than in lovastatin-treated normal fibroblasts. A prominent role for LDL receptors was confirmed by demonstration of similar HTGL-promoted VLDL degradation by normal and LRP-deficient murine embryonic fibroblasts. HTGL enhanced binding and internalization of apoprotein-free triglyceride emulsions, however, this was LDL receptor-independent. HTGL-stimulated binding and internalization of apoprotein-free emulsions was totally abolished by heparinase indicating that it was mediated by HSPG. In a cell-free assay HTGL competitively inhibited the binding of VLDL to immobilized LDL receptors at 4 degrees C suggesting that it may directly bind to LDL receptors but may not bind VLDL particles at the same time.We conclude that the ability of HTGL to enhance VLDL degradation is due to its ability to concentrate lipoprotein particles on HSPG sites on the cell surface leading to LDL receptor-mediated endocytosis and degradation.     

Spin labeled lipid probes in serum lipoproteins

Observations are made relating to the local environment and motional behavior of spin labeled lipids incorporated into human serum lipoproteins. Use has been made of ascorbic acid as a spin subtracting agent to determine the accessibility of the labeled site to the aqueous region.     

Dynamic structure of the lower density lipoproteins. I. Incorporation of high levels of labelled lipids into very low and low density lipoproteins

Selectively labelled lipids have been incorporated into the surface monolayer of human serum low density lipoprotein (LDL) and very low density lipoprotein (VLDL). From 3 to 17 mol% of phosphatidylcholine, selectively deuterated at various positions along the sn-2-acyl chain, was transferred from unilamellar vesicles to VLDL using a partially purified phosphatidylcholine transfer protein. Selectively deuterated palmitic acids were incorporated into LDL (6-20 mol%) and into VLDL (7-10 mol%). Electron microscopy, light scattering, and 31P nuclear magnetic resonance indicated that particle size remained unchanged. Gel exclusion chromatography and chemical analysis showed no difference in hydrodynamic properties and only slight alteration to particle component ratios. Biological activity of labelled VLDL was measured from the rate of cholesterol esterification by cultured J774A.1 cells. Effect of labelling LDL was evaluated by monitoring LDL uptake and degradation by cultured human skin fibroblasts. In all cases the lipoproteins containing labels were indistinguishable from their native counterparts.     

Estimation of surface area in very low density human serum lipoproteins

The surface area of very low density lipoproteins (VLDL) from the serum of 15 healthy donors and the surface area of artificial lipid particles have been estimated. The artificial particles were prepared as a mixture of egg phosphatidylcholine and triolein. Two fluorescent probes - energy donor and acceptor - were placed on the surface, and Forster's nonradiative energy transfer was measured; the transfer efficiency is a function of surface area. The fluorescent probe K-68 (4-[5-(phenyloxazolyl-2)-1-pentadecyl)pyridinium) was used as a donor, and DSP-12 (dimethylamino)styryl-N-dodecylpyridinium) was used as an acceptor. The specific surface area of the artificial lipid particles was estimated to be 0.585 +/- 0.015 nm2 per phosphatidylcholine molecule, which is 15% less than in lipid bilayers. The specific area of VLDL particles was 259 +/- 65 m2 per g of total VLDL. This value is close to the specific area of low density lipoproteins (LDL), and corresponds to the area of a spherical particle 10-12 nm in radius. However, VLDL are assumed to be much larger particles as compared with LDL. Therefore, the new data of the VLDL surface area raise a problem of revision of the existing VLDL models.     

Binding of 5-methoxypsoralen to human serum low density lipoproteins

5-methoxypsoralen (5-MOP) binds to human serum low density lipoproteins (LDL) according to a two-step process. Scatchard analysis of the first step yields K = 1.4 × 10⁵ M^?1 and 4 binding sites. It involves the LDL apoprotein. The second step corresponds to a solubilization, in the lipidic core, of ? 45 molecules of 5MOP per LDL molecule. It is accompanied by a large blue shift of the 5MOP fluorescence. The ability of LDL to bind 5MOP and to carry it into various cells may explain some biological effects sometimes encountered during PUVA therapy.     

Suppression of 3-hydroxy-3-methylglutaryl-CoA reductase by low density lipoproteins produced in vitro by lipoprotein lipase action on nonsuppressive very low density lipoproteins.

Very low density lipoproteins (VLDL), Sf60 to 400, from normolipemic individuals do not suppress 3-hydroxy-3-methylglutaryl-CoA reductase activity in cultured normal human fibroblasts at concentrations 20-fold higher than those of low density lipoproteins (LDL) that give total suppression. To determine if these VLDL contain all of the structural elements necessary for receptor-mediated suppression, they were converted in vitro with bovine milk lipoprotein lipase to low density lipoproteins. These LDL-like lipoproteins were as effective in suppression as LDL isolated directly from plasma, with half-maximal and complete suppression at 1 and 4 microgram of cholesterol ml-1. Neither native LDL nor LDL produced in vitro suppressed receptor-negative fibroblasts. We conclude that action of lipoprotein lipase on VLDL leads to a rearrangement of lipoprotein components that permits interaction of LDL produced in vitro with the LDL-specific cell surface receptor of fibroblasts and subsequent suppression of 3-hydroxy-3-methylglutaryl-CoA reductase.