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Characterization of HDL and lipoproteins intermediate to LDL and HDL in the serum of pedigreed baboons fed an atherogenic diet.

A lipoprotein species with ultracentrifugal flotation rates (F0(1.20) 9-28) intermediate to high density lipoproteins (HDL, F0(1.20) 0-9) and low density lipoproteins (LDL, F0(1.20) 28-56) found in the plasma of certain pedigreed baboons fed an atherogenic diet was studied by gradient gel electrophoresis (GGE) and ultracentrifugal techniques. These lipoproteins were found to be heterogeneous in size (125-220 A) and hydrated density (1.028-1.080 g/ml). The major apolipoprotein in all density subfractions of the F0(1.20) 9-28 lipoproteins exhibited the molecular weight (2.8 X 10(4) daltons) and immunochemical properties of apolipoprotein A-I (apoA-I). Protein corresponding to apolipoprotein E (apoE, 3.5 X 10(4) daltons) was observed primarily in the less dense subspecies of F0(1.20) 9-28 lipoproteins. Some low molecular weight (1.8 X 10(4), 1.3 X 10(4), and 1.1 X 10(4) daltons) apolipoproteins were also detected. At low serum F0(1.20) 9-28 lipoprotein concentrations, only the smaller, more dense, protein-rich species were present; at higher F0(1.20) 9-28 concentrations, the larger, less dense species were observed in addition to the small species. The HDL of pedigreed baboons in families with and without serum F0(1.20) 9-28 lipoproteins were also characterized. The HDL of both groups of progeny consisted of a similar set of 5 subpopulations designated HDL-I through HDL-V determined by GGE. HDL-I, consisting of material 100-125 A in size, was the major HDL subpopulation. ApoA-I was the major protein moiety in all HDL subpopulations; none contained apoE. Baboons in families with F0(1.20) 9-28 lipoproteins had more HLD-I (292 +/- 80 mg/dl vs. 235 +/- 55 mg/dl) and less HDL-II (86 +/- 22 mg/dl vs. 135 +/- 34 mg/dl) than baboons in families without F0(1.20) 9-28 lipoproteins; both groups showed identical total HDL concentrations (446 +/- 90 mg/dl and 444 +/- 49 mg/dl, respectively). Among those baboons in families with F0(1.20) 9-28 lipoproteins, there was an inverse correlation between F0(1.20) 9-28 concentration and total HDL, HDL-I and HDL-II concentrations, indicating a possible metabolic relationship between these HDL subpopulations and the F0(1.20) 9-28 species.

Animals↗

Small polydisperse low density lipoproteins in familial hyperalphalipoproteinemia with complete deficiency of cholesteryl ester transfer activity.

Lipoprotein abnormalities were analyzed in 3 cases of marked hyperalphalipoproteinemia caused by complete deficiency of cholesteryl ester transfer activity. The probands were all men, aged 34, 43 and 48 years, respectively. The serum high density lipoprotein (HDL)-cholesterol levels of these patients were higher than 150 mg/dl (157-254 mg/dl), while serum total cholesterol levels ranged from 227 to 360 mg/dl. Sequential flotation-ultracentrifugation analysis disclosed that low density lipoprotein (LDL)-cholesterol was slightly decreased and that cholesteryl ester accumulated solely in the HDL2 fraction, which was also enriched with apolipoprotein E. Cholesteryl ester transfer activity was completely absent in all of these cases. High-performance liquid chromatography showed a decrease of LDL particle size in combination with a marked enlargement of HDL particle size. Analytical ultracentrifugation disclosed heterogeneity of LDL with the presence of small LDL subpopulations. We conclude that hyperalphalipoproteinemia due to complete deficiency of cholesteryl ester transfer activity is characterized by the presence of both small polydisperse LDL and markedly large HDL enriched with cholesteryl ester and apolipoprotein E.

Adult↗

Characteristics of low density lipoprotein isolated from circulating immune complexes.

Circulating immune complexes (CIC) containing low density lipoprotein (LDL) were recently found in the blood of patients with coronary atherosclerosis. In the present study, we investigated the chemical composition and physical characteristics of the lipoprotein constituents of these CIC. CIC were isolated from the blood of atherosclerotic patients by affinity chromatography using anti-human immunoglobulin G-agarose. Low density lipoprotein of these complexes (CIC-LDL) was obtained by ultracentrifugation. CIC-LDL was compared with free circulating LDL isolated from the blood plasma of the same patients. Plasma LDL was fractionated by lectin-chromatography on RCA120-agarose to obtain desialylated LDL (atherogenic) and sialylated LDL (nonatherogenic). Both CIC-LDL and desialylated LDL, but not native (sialylated) lipoprotein, induced a 1.8- to 3-fold increase in the intracellular contents of free and esterified cholesterol of cells cultured from grossly normal areas of human aorta. The sialic acid level in CIC-LDL was 1.3- and 2.1-fold lower than in desialylated or native LDL, respectively. The neutral lipid and phospholipid contents of CIC-LDL and desialylated LDL were reduced as compared to native LDL. The levels of lipid-oxidation products, thiobarbituric acid-reactive substances and hydroperoxides, were similar in all lipoprotein preparations. However, desialylated LDL and CIC-LDL had an elevated oxysterol content. Gradient ultracentrifugation revealed that CIC-LDL particles had a higher density than native LDL. The mean diameters of native, desialylated and CIC-LDL accounted for 24.0, 21.3 and 19.5 nm, respectively. Like desialylated LDL, CIC-LDL displayed a higher electrophoretic mobility compared with that of native LDL. Thus, LDL obtained from circulating immune complexes appears to be a multiple-modified lipoprotein possessing many similarities to desialylated LDL. It was also found that the LDL content of circulating immune complexes correlates well with the desialylated LDL level in human plasma but not with the total LDL concentration. We believe that desialylated LDL predominately interacts with antibodies forming immune complexes. Taken together, our findings suggest that multiple-modified desialylated LDL is the circulating autoantigen for anti-LDL autoantibodies.

Adult↗

Ferric acetate-uranium acetate reagent in cholesterol estimations in plasma and high density lipoprotein fractions: comparison with existing methods.

The use of ferric acetate-uranium acetate colour reaction for the estimation of cholesterol in the supernatants of plasma samples after precipitation of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol by heparin-MnCl2 was assessed and compared with the conventional method using the FeCl3 colour reaction and also with the method using o-phthalaldehyde as the colouring reagent. All three methods gave comparable values when total cholesterol in plasma samples was determined and also when high density lipoprotein (HDL) fractions were separated by ultracentrifugation and the cholesterol contents determined. But when heparin-MnCl2 precipitation was used for HDL separation, and the cholesterol content determined, the FeCl3 method gave significantly lower values. This could be due to interference of the cholesterol colour reaction with FeCl3, due to Mn2+ ions present in the supernatant. Addition of Mn2+ to cholesterol standards and subsequent colour development with ferric acetate-uranium acetate and FeCl3 reagents showed that Mn2+ decreased the absorbancy of the coloured complex at 560 nm only when FeCl3 was used. Percentage recovery of added cholesterol was also lower when the heparin-MnCl2 supernatant was treated with FeCl3 reagent for colour development. Use of ferric acetate-uranium acetate reagent provides a simpler and quicker method. It does not suffer from interference due to the presence of Mn2+ ions and gives results comparable to the o-phthalaldehyde method and those using ultracentrifugation as the separation procedure.

Chemical Precipitation↗

Isolation of pig Clq and its structural and functional characterization.

Pig Clq, a subcomponent of the first component of complement, was isolated in a fully hemolytically active form using precipitation in the presence of chelating agents at low ionic strength. Three times precipitated Clq was highly purified as shown by immunoelectrophoresis, polyacrylamide gel electrophoresis and ultracentrifugation. The yield of Clq using this method ranged from 40 to 55%. Pig Clq was also purified using precipitation with EDTA followed by affinity chromatography on a pig IgG-Sepharose 4B column. However, overall recovery of Clq was only about 10%. The purified Clq was heat-labile (56 degrees C, 30 min) both structurally and functionally, had an apparent molecular weight of 400 600 daltons as determined by ultracentrifugation, and restored the hemolytic complement activity of Clq-depleted sera of various species thus confirming interchangeability of this subcomponent. Cross-reactivity of pig, human, guinea-pig, mouse, rat and frog Clq-containing sera with monospecific anti-pig Clq antiserum showed a high degree of antigenic similarity.

Animals↗

Effect of Sendai inducing virus in cytopathic effect inhibition assay of bovine leukocyte interferon.

Various methods for removal or inactivation of Sendai inducing virus from natural bovine leukocyte interferon (IFN) preparations were compared and the effects of Sendai virus in microtiter cytopathic effect (CPE) inhibition assay were investigated. Ultraviolet light and ultracentrifugation at either 108,000 X g or 145,000 X g for 2 h proved to be inadequate methods for complete removal of inducing virus from IFN preparations, as judged by the effects of residual virus in CPE inhibition assay. Optimal methods for inducer virus removal/inactivation were ultracentrifugation at 175,000 X g or dialysis against pH 2.0 buffer. Sendai virus itself was capable of inducing IFN production in assay cells at concentrations commonly found in virus-induced IFN preparations.

Animals↗

Rapid virus subunit visualization by direct sedimentation of samples on electron microscope grids.

Airfuge direct ultracentrifugation of viral samples on electron microscope grids offers a rapid way for concentrating viral particles or subunits to facilitate their detection and study. Using the A-100 fixed angle rotor (30 degrees) with a K factor of 19 at maximum speed (95,000 rpm), samples up to 240 microliters can be prepared for electron microscopy observation in a few minutes: observation time is decreased and structural details are highlighted. Using latex spheres to calculate the increase in sensitivity compared to the inverted drop procedure, we obtained a 10- to 40-fold increase in sensitivity depending on the size of particles. Application of this technique to rubella virus permitted better visualization of viral membrane subunits on the particles. Rubella hemagglutinin immuno-stimulating complexes preparations were also better visualized and their morphology conserved after direct ultracentrifugation on the specimen grids. Similar observations are reported for respiratory syncytial virus associated subunits.

Animals↗

Rapid method for the isolation of two purified subfractions of high density lipoproteins by differential dextran sulfate-magnesium chloride precipitation.

We describe a rapid and reliable three-step precipitation procedure for the isolation of large amounts of the two major components of high density lipoproteins (HDL) in human serum. Precipitation was accomplished by means of dextran sulfate (DS) of mol. wt. 500,000 and MgCl2. First, all apoB-associated lipoproteins of any density were selectively precipitated with critical concentrations of reagents. Secondly, a subfraction of HDL was differentially precipitated from the apoB-depleted serum by increasing the concentration of both reagents. Eventually, the bulk of the remainder of HDL was precipitated by lowering the pH to 5.4. According to the precipitation patterns and the density profiles, the DS-Mg procedure provides a clear differentiation between the two HDL components. According to the compositional criteria and the ultracentrifugal characteristics, the two polyanion-precipitated subclasses are very similar to, if not identical with, the two density subclasses, the lighter HDL2 and the heavier HDL3, isolated by preparative ultracentrifugation after apoB-containing lipoproteins had been removed.

Chemical Precipitation↗

Biochemical studies of taste sensation. XI. Isolation, characterization and taste ligand binding activity of plasma membranes from catfish taste tissue.

Plasma membranes were isolated from taste receptor-containing epithelium of the channel catfish, Ictalurus punctatus. The membranes were prepared by ultracentrifugation of a sedimentable fraction in sucrose, using either a discontinuous density gradient or a continuous linear density gradient. The plasma membranes were characterized by their increased content of 5'-nucleotidase and by electron microscopy, as well as by a greatly diminished content of NADH-cytochrome c reductase and succinate-cytochrome c reductase. The recovery of binding activity for taste ligands was low, because of the long time-period required for ultracentrifugation, but of the recovered activity 80% occurred in the plasma-membrane preparation. Binding of seven chemostimulatory amino acids was demonstrated and found to correspond reasonably well with earlier binding data obtained using a less pure sedimentable fraction. The data provide direct evidence supporting the long-standing hypothesis that taste receptor sites are localized to the plasma membranes.

Amino Acids↗

Comparison of different methods for LDL isolation and radioiodination on liver LDL receptor binding in vitro.

Lipoproteins were isolated either by immunoaffinity chromatography (LDL and VLDL) or ultracentrifugation (LDL). Purified lipoproteins were labeled with 123I using either Iodogen or iodine-monochloride (IC1) each followed by purification with gel-chromatography or dialysis (total of 4 combinations). Lipoprotein-concentrations of 0.1-6 micrograms protein/mL were used for direct binding assays investigating the specific binding of labeled lipoproteins (in the presence of a 50-fold excess of unlabeled lipoproteins) to human liver apo-B, E-receptors. In separate experiments displacement of bound 123I-lipoproteins (labeled by the methods mentioned) by unlabeled ones was studied. The binding capacities estimated by Scatchard analysis were similar to each other (141-163 ng protein bound/mg liver plasma membrane protein) independent from the method used for isolation and labeling. Also the affinity constants were very similar and ranged from 0.9 to 1.7 micrograms protein/L. It is concluded that immunoaffinity chromatography or ultracentrifugation for isolation of lipoproteins and the Iodogen or IC1-method for radiolabeling can be recommended to be equally good for in vitro receptor investigation.

Adult↗

A model for sedimentation in inhomogeneous media. II. Compressibility of aqueous and organic solvents.

The effects of solvent compressibility on the sedimentation behavior of macromolecules as observed in analytical ultracentrifugation are examined. Expressions for the density and pressure distributions in the solution column are derived and combined with the finite element solution of the Lamm equation in inhomogeneous media to predict the macromolecular concentration distributions under different conditions. Independently, analytical expressions are derived for the sedimentation of non-diffusing particles in the limit of low compressibility. Both models are quantitatively consistent and predict solvent compressibility to result in a reduction of the sedimentation rate along the solution column and a continuous accumulation of solutes in the plateau region. For both organic and aqueous solvents, the calculated deviations from the sedimentation in incompressible media can be very large and substantially above the measurement error. Assuming conventional configurations used for sedimentation velocity experiments in analytical ultracentrifugation, neglect of the compressibility of water leads to systematic errors underestimating sedimentation coefficients by approximately 1% at a rotor speeds of 45000 rpm, but increasing to 2-5% with increasing rotor speeds and decreasing macromolecular size. The proposed finite element solution of the Lamm equation can be used to take solvent compressibility quantitatively into account in direct boundary models for discrete species, sedimentation coefficient distributions or molar mass distributions. Using the analytical expressions for the sedimentation of non-diffusing particles, the ls-g*(s) distribution of apparent sedimentation coefficients is extended to the analysis of sedimentation in compressible solvents. The consideration of solvent compressibility is highly relevant not only when using organic solvents, but also in aqueous solvents when precise sedimentation coefficients are needed, for example, for hydrodynamic modeling.

Centrifugation, Density Gradient↗

Distribution of apolipoprotein E among lipoprotein fractions in the lactating cow.

Apolipoprotein (apo) E plays a key role in regulating plasma levels of lipoproteins. We investigated the serum apoE concentrations in cows during different lactating stages by ELISA. To confirm the distribution of apoE in lipoprotein fractions, cow plasma was separated by gel filtration, ultracentrifugation and agarose gel electrophoresis. The apoE concentrations during early, mid- and late lactating stages in cows were significantly higher than that during the non-lactating stage. In lactating plasma, apoE eluted in high-density lipoprotein (HDL) fractions separated by gel filtration increased. The portion of this apoE in plasma was 49%. However, when lactating plasma was separated by ultracentrifugation, less then 5% apoE was recovered in the HDL fraction, and more apoE was recovered in the non-lipoprotein fraction (d>1.21 g/ml, 46%). In agarose gel electrophoresis, plasma apoE was found in beta-migrating lipoprotein, but it was not present in alpha-migrating lipoprotein. To purify apoE-containing particles, the HDL fraction separated by gel filtration was pooled and the fraction retained on Heparin-Sepharose chromatography collected. Cholesterol was absent from this fraction. These results suggest that apoE-containing particles, which increased during the lactating stage, were not associated with HDL particles, and that lipid-free forms were included in cow plasma.

Animals↗

A practical approach to optimization and validation of a HPLC assay for analysis of polyribosyl-ribitol phosphate in complex combination vaccines.

The use of multi-factor statistical experimental design methodology minimized the vaccine material and laboratory resources required for optimization and validation of an HPLC assay for quantitation of depolymerized and total PRP. Components of the assay selected for optimization were adjuvant dissolution, ultracentrifuge conditions including ultracentrifuge model, sample diluent, mobile phase and column oven temperature. Previous experience has shown these components of the assay to be most troublesome and therefore required optimization prior to validation. Specificity, linearity, precision, accuracy and ruggedness were confirmed through a validation of the optimized assay. The validation also established the assay to be stability indicating, by showing that changes to the integrity of the PRP-OMPC conjugate could be detected.

Chromatography, High Pressure Liquid↗

Association of Lp(a) rather than integrally-bound apo(a) with triglyceride-rich lipoproteins of human subjects.

The majority of apolipoprotein (a) [apo(a)] in plasma is characteristically associated with Lipoprotein (a) [Lp(a)], having a buoyant density (1.05-1.08 g/ml) intermediate between low density lipoproteins (LDL) and high density lipoproteins (HDL). In the fed (postprandial) state or in the presence of fasting (endogenous) hypertriglyceridemia, a small proportion of plasma apo(a) is found in the density < 1.006 g/ml fraction of plasma, associated with larger and less dense triglyceride-rich lipoproteins (TRL). In order to further characterize the presence of apo(a) in ultracentrifugally-separated TRL (UTC-TRL), this lipoprotein fraction was isolated from plasma obtained in the fed state (three hours after an oral fat load) from healthy normolipidemic subjects (Lp(a): 38 +/- 8 mg/dl (mean +/- S.E.), n = 4) and also from plasma obtained after an overnight fast from hypertriglyceridemic patients (plasma TG: 8.16 +/- 2.00 mmol/l, Lp(a): 41 +/- 3 mg/dl, n = 18). Apo(a) in 3 h-postprandial UTC-TRL (5 +/- 2% of total plasma apo(a)) and in hypertriglyceridemic UTC-TRL (8 +/- 2% total apo(a)) was separable by electrophoresis and/or gel chromatography (FPLC) from the majority of UTC-TRL lipid. Apo(a) in UTC-TRL fractions had slow pre-beta electrophoretic mobility and was isolated in a lipoprotein size-range smaller than VLDL and larger than LDL, consistent with it being Lp(a). Recentrifugation of UTC-TRL resulted in the majority of apo(a) being recovered in the density > 1.006 g/ml fraction. Addition of proline to plasma samples before ultracentrifugation (final concentration: 0.1 M) substantially reduced the amount of Lp(a) in UTC-TRL. TRL separated from plasma by FPLC contained less apo(a) (2-5% of total plasma apo(a)), but this apo(a) was also readily dissociable from TRL lipid, had slow pre-beta electrophoretic mobility, and was associated with a lipoprotein with the size of Lp(a). Our data suggest that apo(a) in the TRL fraction of subjects with postprandial triglyceridemia or endogenous hypertriglyceridemia is not an integral component of plasma VLDL or chylomicrons, but represents the presence of non-covalently bound Lp(a).

Adult↗

Biophysical characterization of the influence of salt on tetrameric SecB.

SecB is a tetrameric chaperone, with a monomeric molecular mass of 17 kDa, that is involved in protein translocation in Escherichia coli. It has been hypothesized that SecB undergoes a conformational change as a function of the salt concentration. To gain more insight into the salt-dependent behavior of SecB, we studied the protein in solution by dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, and small angle neutron scattering. The results clearly demonstrate the large influence of the salt concentration on the behavior of SecB. At high salt concentration, SecB is a non-spherical protein with a radius of gyration of 3.4 nm. At low salt concentration the hydrodynamic radius of the protein is apparently decreased, whereas the ratio of the frictional coefficients is increased. The protein solution behaves in a non-ideal way at low salt concentrations, as was shown by the analytical ultracentrifugation data and a pronounced interparticle effect observed by small angle neutron scattering. A possible explanation is a change in surface charge distribution dependent on the salt concentration in the solvent. We summarize our data in a model for the salt-dependent conformation of tetrameric SecB.

Bacterial Proteins↗

Zone centrifugation in a cesium chloride density gradient caused by temperature change.

In this communication is described a new technique for the determination of sedimentation coefficients of macromolecules banded in equilibrium density gradients. Initially, the macromolecules are banded in the analytical ultracentrifuge at a low temperature of about 5 degrees C. After equilibrium has been obtained, the temperature is increased to 25 degrees C. The equilibrium band will now sediment to a new equilibrium position in the ultracentrifuge cell: (a) By following the position of the migrating band as a function of time, sedimentation coefficients may be determined. (b) If several species having different sedimentation coefficients are present in the original band, then during the course of the migration the band may split into several new bands which eventually reunite at the final equilibrium position. (c) If different chemical species of macromolecules such as nucleic acids and carbohydrates are present, in general they will exhibit different temperature density relationships, and can move different distances and directions in response to temperature change.

Carbohydrates↗

High-resolution, submicron particle size distribution analysis using gravitational-sweep sedimentation.

Sedimentation velocity is a powerful tool for the analysis of complex solutions of macromolecules. However, sample turbidity imposes an upper limit to the size of molecular complexes currently amenable to such analysis. Furthermore, the breadth of the particle size distribution, combined with possible variations in the density of different particles, makes it difficult to analyze extremely complex mixtures. These same problems are faced in the polymer industry, where dispersions of latices, pigments, lacquers, and emulsions must be characterized. There is a rich history of methods developed for the polymer industry finding use in the biochemical sciences. Two such methods are presented. These use analytical ultracentrifugation to determine the density and size distributions for submicron-sized particles. Both methods rely on Stokes' equations to estimate particle size and density, whereas turbidity, corrected using Mie's theory, provides the concentration measurement. The first method uses the sedimentation time in dispersion media of different densities to evaluate the particle density and size distribution. This method works provided the sample is chemically homogeneous. The second method splices together data gathered at different sample concentrations, thus permitting the high-resolution determination of the size distribution of particle diameters ranging from 10 to 3000 nm. By increasing the rotor speed exponentially from 0 to 40,000 rpm over a 1-h period, size distributions may be measured for extremely broadly distributed dispersions. Presented here is a short history of particle size distribution analysis using the ultracentrifuge, along with a description of the newest experimental methods. Several applications of the methods are provided that demonstrate the breadth of its utility, including extensions to samples containing nonspherical and chromophoric particles.

Deuterium Oxide↗

Hydrodynamic properties of human erythrocyte band 3 solubilized in reduced Triton X-100.

The oligomeric state and function of band 3, purified by sulfhydryl affinity chromatography in reduced Triton X-100, was investigated. Size exclusion high-performance liquid chromatography showed that a homogeneous population of band 3 dimers could be purified from whole erythrocyte membranes. The elution profile of band 3 purified from membranes that had been stripped of its cytoskeleton before solubilization was a broad single peak describing a heterogeneous population of oligomers with a mean Stokes radius of 100 A. Sedimentation velocity ultracentrifugation analysis confirmed particle heterogeneity and further showed monomer/dimer/tetramer equilibrium self-association. Whether the conversion of dimer to the form described by a Stokes radius of 100 A was initiated by removal of cytoskeletal components, alkali-induced changes in band 3 conformation, or alkali-induced loss of copurifying ligands remains unclear. After incubation at 20 degrees C for 24 h, both preparations of band 3 converted to a common form characterized by a mean Stokes radius of 114 A. This form of the protein, examined by equilibrium sedimentation ultracentrifugation, is able to self-associate reversibly, and the self-association can be described by a dimer/tetramer/hexamer model, although the presence of higher oligomers cannot be discounted. The ability of the different forms of the protein to bind stilbene disulfonates revealed that the dimer had the highest inhibitor binding affinity, and the form characterized by a mean Stokes radius of 114 A to have the lowest.

Anion Exchange Protein 1, Erythrocyte↗