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.     

Crystallization effects in serum high density lipoproteins

Peculiarities of aggregation in the samples of high density serum lipoproteins LHD2 and LHD3 obtained from healthy donors and patients with ishaemic heart disease were studied under isothermal exposition. It was found that lipoprotein samples from defective serum were characterized by chemical and structural inhomogeneity. It is reflected in the formation of spherolites of various types and of well-grown dendrite forms. Growth of stepped lens-shaped crystals of cholesterol takes place in the normal serum LHD3 preparations after long exposition. Crystal growth is thought to begin according to dislocational mechanism on linear defects existing in the liquid crystalline phase; then transformation to kinetically rough surface and to normal growth of the face occurs with the concentration increase.     

Properties of rooster serum high density lipoproteins.

1. Holo-superoxide dismutase from bovine erythrocytes has been shown to undergo a reversible structural modification in the pH 3-5 range. 2. The spectral alterations observed on changing from neutrality to pH 2 were: a slight attenuation of the 680 nm absorbance; the loss of the 450 nm shoulder, apparent in the optical spectrum of the native protein; and a new band appeared at 330 nm. The circular dichroism at 600 nm was essentially lost while a weak negative band appeared at approx. 380 nm and a positive band at 310 nm. 3. The EPR spectrum was also modified on changing from the native to the low pH form: A parallel increased from approximately 130 to approximately 150 G, g parallel remained unchanged at approximately 2.27, and gm decreased from approximately 2.09 to approximately 2.08. The apparent linewidth remained essentially constant. 4. High resolution (220 MHz) PMR spectra of holo- and apoproteins revealed that the metals influence the three-dimensional structure of the protein. 5. PMR studies indicated that at pH 3 the apoprotein existed almost entirely in a random coil form and that it assumed a compact well-ordered structure on returning to neutral pH. The holoprotein maintained a compact, apparently dimeric, structure even at pH 3.     

Subunit structure of the apoprotein of human serum low density lipoproteins

Human serum low density lipoproteins (d 1.027–1.043 g/cm³) were prepared by preparative ultracentrifugation and delipidated with sodium deoxycholate. By electrophoresis in sodium dodecyl sulfate polyacrylamide gel, the apoprotein was fractionated into major components with apparent molecular weights of 77,000, 66,000, 47,000, 33,500, 21,500, 13,000, and 9,500, respectively; and minor components of higher molecular weight. The data indicate the existence of at least two fundamental subunits of molecular weights of approximately 9,500 and 13,000 daltons.     

Particle distribution of human serum high density lipoproteins.

Density gradient ultracentrifugation of human serum high density lipoproteins (HDL) from both normolipemic males and females results in a distribution of HDL concentration versus subfraction hydrated density which has three maxima. Gradient gel electrophoresis of total HDL is characterized by three banding maxima, the positions of which suggest the presence of three particle size ranges: I. 10.8-12.0 nm, II. 9.7-10.7 nm, and III. 8.5-9.6 nm. Gradient gel electrophoresis of density gradient subfractions established an inverse relationship between particle size and particle hydrated density which was corroborated by electron microscopy and analytic ultracentrifugation. Comparison of male HDL from size ranges I, II, and III with female HDL from the same size ranges showed only small differences in the mean value of the peak F degrees 1.20 rate, size, molecular weight, protein weight percent, and weight protein/weight phospholipid. Major differences between males and females were seen in the relative amounts of HDL in density gradient subfractions 1-3 (size range I material) and 11-12 (size range III material); the percent total HDL in the group of subfractions 1-3 was greatly increased in female HDL while that of the group of subfractions 11-12 was increased in the male HDL. These studies indicate the presence of at least three major components in HDL instead of two (HDL2 and HDL3) and that peak F degrees 1.20 rate differences in HDL schlieren patterns between males and females are a function of the relative levels of these three components.     

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.     

Incorporation of excess cholesterol by high density serum lipoproteins.

Excess cholesterol was added to human HDL3 and to bovine mammalian high density serum lipoprotein (HDL) by incubating aqueous lipoprotein solutions with solid dispersions of [4-(14)C]cholesterol on Celite. Lipoprotein cholesterol complexes were isolated by centrifugation and filtration through a Sepharose 4B column. The pure complexes were analyzed for protein and lipid content and composition and were subsequently investigated by physical methods (analytical ultracentrifugation, circular dichroism, and fluorescence spectroscopy), in order to detect any structural changes induced by added cholesterol. The rates of cholesterol uptake varied as an inverse function of the intrinsic cholesterol present in the native lipoproteins. The maximum cholesterol taken up by human HDL3 increased the free cholesterol content from 3--4% (initial) up to 22% of the total lipoprotein weight. Bovine HDL was observed to increase its free cholesterol content from 2--4% (initial) up to 11--17% of the total lipoprotein weight, before denaturation. At maximum levels of added cholesterol, both lipoproteins had increased molecular weights and sedimentation velocity coefficients corresponding to the increased mass of the particles. No major changes in the hydrodynamic properties were observed. At the molecular level, the protein components only showed a 15--20% decrease in fluorescence intensity, possibly a consequence of a modified environment of the aromatic amino acid residues. In the human HDL3, added cholesterol increased the microviscosity of the lipid domains by 1.2 P at 25 degrees C (from 3.4 to 4.6 P), but did not affect the fluidity of bovine HDL lipids (5.9 P).     

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.     

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.     

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.     

Lipid transfer between human serum high density lipoproteins and egg yolk lipoproteins in incubation mixtures

Ultracentrifugally isolated human serum high density lipoproteins of d 1.063-1.21 (HDL) were incubated with egg yolk lipoproteins of d < 1.006 for up to 24 hr at various concentrations. Transfer of HDL cholesterol esters to egg yolk lipoproteins occurred simultaneously with transfer of glycerides from egg yolk lipoproteins to HDL. These observations show that exchange of lipids can take place between lipoproteins in the absence of other serum proteins and enzymes. The mole ratios of HDL cholesterol esters to glycerides approached an integral value of 1 : 1 during the course of the incubation. These results suggest that lipid components form complexes within the HDL structure.     

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.