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PubMed1993

Low density lipoprotein subfractions and [Ca2+]i in vascular smooth muscle cells.

Several studies have established that plasma low density lipoprotein (LDL) consists of various discrete subfractions. Using a variety of techniques (analytical ultracentrifugation, equilibrium density gradient ultracentrifugation, and gradient gel electrophoresis), LDL has been fractionated into a maximum of seven subclasses that differ in particle size, density, and physiochemical composition. Recently, a predominance of smaller denser LDL particles has been associated with an increased risk of coronary artery disease. However, other lipoprotein changes, such as elevated triglycerides and lower HDL cholesterol levels, have been shown in patients with a predominance of the smaller denser LDL subfractions. Thus, it is unclear whether the enhanced atherogenic potential is induced by the LDL subfraction pattern per se or by concomitant lipoprotein changes. Because intracellular free Ca2+ is an important second messenger involved in atherogenesis and regulation of vascular tone, we studied the influence of three LDL subfractions (very light [LDL1], 1.030 to 1.033 g/mL; light [LDL2], 1.033 to 1.040 g/mL; and dense [LDL3], 1.040 to 1.045 g/mL) on [Ca2+]i in vascular smooth muscle cells (VSMCs) cultured from rat aorta. LDL subfractions were isolated by density gradient ultracentrifugation from human EDTA-plasma (n = 15). [Ca2+]i was measured by fura 2 fluorescence. Basal [Ca2+]i was 77 +/- 6 nmol/L. Stimulation of VSMCs with dense LDL3 caused a significantly (P < .05) more pronounced increase (+71 +/- 13 nmol/L) compared with LDL1 (+38 +/- 8 nmol/L) and LDL2 (+36 +/- 9 nmol/L). To further investigate the mechanisms leading to the stimulation of [Ca2+]i by LDL subfractions, we incubated VSMCs with the Ca2+ antagonists nifedipine, diltiazem, and verapamil in concentrations up to 10 mumol/L.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1982

Lipoprotein metabolism during acute inhibition of hepatic triglyceride lipase in the cynomolgus monkey.

The role of the enzyme hepatic triglyceride lipase was investigated in a primate model, the cynomolgus monkey. Antisera produced against human postheparin hepatic lipase fully inhibited cynomolgus monkey posttheparin plasma hepatic triglyceride lipase activity. Lipoprotein lipase activity was not inhibited by this antisera. Hepatic triglyceride lipase activity in liver biopsies was decreased by 65-90% after intravenous infusion of this antisera into the cynomolgus monkey. After a 3-h infusion of the antisera, analytic ultracentrifugation revealed an increase in mass of very low density lipoproteins (S(f) 20-400). Very low density lipoprotein triglyceride isolated by isopycnic ultracentrifugation increased by 60-300%. Analytic ultracentrifugation revealed an increase in mass of lipoproteins with flotation greater than S(f) 9 (n = 4). The total mass of intermediate density lipoproteins (S(f) 12-20) approximately doubled during the 3 h of in vivo enzyme inhibition. While more rapidly floating low density lipoproteins (S(f) 9-12) increased, the total mass of low density lipoproteins decreased after infusion of the antibodies. The changes in high density lipoproteins did not differ from those in control experiments. In order to determine whether the increases of plasma concentrations of very low density lipoproteins were due to an increase in the rate of synthesis or a decrease in the rate of clearance of these particles, the metabolism of radiolabeled homologous very low density lipoproteins was studied during intravenous infusion of immunoglobulin G prepared from the antisera against hepatic triglyceride lipase (n = 3) or preimmune goat sera (n = 3). Studies performed in the same animals during saline infusion were used as controls for each immunoglobulin infusion. There was a twofold increase in the apparent half-life of the very low density lipoprotein apolipoprotein-B tracer in animals receiving the antibody, consistent with a decreased catabolism of very low density lipoproteins. Concomitantly, the rise in low density lipoprotein apoprotein-B specific activity was markedly delayed. None of these changes were observed during infusion of preimmune immunoglobulin G.Hepatic triglyceride lipase participates with lipoprotein lipase in the hydrolysis of the lipid in very low density lipoproteins, intermediate density lipoproteins, and the larger low density lipoproteins (S(f) 9-12). Thus, hepatic triglyceride lipase appears to function in a parallel role with lipoprotein lipase in the conversion of very low density and intermediate density lipoproteins to low density lipoproteins (S(f) 0-9).

Animals↗

PubMed2004

Association of a model transmembrane peptide containing gly in a heptad sequence motif.

A peptide containing glycine at a and d positions of a heptad motif was synthesized to investigate the possibility that membrane-soluble peptides with a Gly-based, left-handed helical packing motif would associate. Based on analytical ultracentrifugation in C14-betaine detergent micelles, the peptide did associate in a monomer-dimer equilibrium, although the association constant was significantly less than that reported for the right-handed dimer of the glycophorin A transmembrane peptide in similar detergents. Fluorescence resonance energy transfer (FRET) experiments conducted on peptides labeled at their N-termini with either tetramethylrhodamine (TMR) or 7-nitrobenz-2-oxa-1,3-diazole (NBD) also indicated association. However, analysis of the FRET data using the usual assumption of complete quenching for NBD-TMR pairs in the dimer could not be quantitatively reconciled with the analytical ultracentrifugation-measured dimerization constant. This led us to develop a general treatment for the association of helices to either parallel or antiparallel structures of any aggregation state. Applying this treatment to the FRET data, constraining the dimerization constant to be within experimental uncertainty of that measured by analytical ultracentrifugation, we found the data could be well described by a monomer-dimer equilibrium with only partial quenching of the dimer, suggesting that the helices are most probably antiparallel. These results also suggest that a left-handed Gly heptad repeat motif can drive membrane helix association, but the affinity is likely to be less strong than the previously reported right-handed motif described for glycophorin A.

Amino Acid Motifs↗

PubMed1995

Changes in the concentration and distribution of lipoprotein (a) in plasma after fat intake.

To clarify the association between apo(a) and TG-rich lipoproteins, we studied changes in plasma Lp(a) concentration and apo(a) distribution in lipoprotein fractions after fat intake. The subjects were 15 hyperlipidemic patients and 3 healthy volunteers with fasting Lp(a) concentrations ranging from 2.5-52 mg/dl. They were given a fatty meal (50 g fat/m2 and 60 mg retinyl palmitate) after a 12-hour overnight fast and venous blood samples were taken at 0, 3, 4.5, 6, 7.5, 9, 12 and 24 hours. Fractions of sf > 400, sf 20-400 and d > 1.006 g/ml were isolated from plasma samples by ultracentrifugation. Plasma Lp(a) levels increased transiently at 4.5 hours, decreased between 4.5 and 12 hours, and recovered almost to initial levels by the next morning. Plasma Lp(a) peaked before the plasma RP peak appeared. Apo(a) associated with TG-rich lipoprotein fractions increased and apo(a) in the d > 1.006 fraction decreased after fat intake in most of the subjects, suggesting the transfer of apo(a) from the d > 1.006 fraction to the TG-rich lipoproteins. The increase in TG-rich lipoprotein apo(a) correlated with the RP area under the curve (r=0.79, p<0.05) and the decrease in d > 1.006 apo(a) (r = 0.80, p < 0.05). This distributional change of apo(a) after fat intake was confirmed by gel filtration and density gradient ultracentrifugation. Transfer of apo(a) from the main Lp(a) fraction of the plasma obtained from a subject with a high Lp(a)level to the TG-rich lipoprotein fraction of the plasma obtained 4.5 hours after fat intake from a subject with a low plasma Lp(a) level was also shown in vitro by density gradient ultracentrifugation. Our studies revealed a significant association between apo(a) and TG-rich lipoproteins in the postprandial. Further studies are necessary to clarify the pathophysiological role of Lp(a) in TG-rich lipoproteins.

Adult↗

PubMed2005

Advanced lipoprotein testing does not improve identification of subclinical atherosclerosis in young adults: the Bogalusa Heart Study.

BACKGROUND: The clinical value of advanced lipoprotein testing relative to traditional lipid testing remains controversial. To date, no studies have evaluated associations between advanced lipoprotein testing and subclinical atherosclerosis in healthy young adults. OBJECTIVE: To determine whether advanced lipoprotein testing using vertical-spin density-gradient ultracentrifugation better predicts carotid intima-media thickness, a validated measure of subclinical atherosclerosis, than does traditional lipoprotein testing in asymptomatic young adults. DESIGN: Cross-sectional community-based study. SETTING: Bogalusa, Louisiana. PARTICIPANTS: 311 randomly selected adults from the Bogalusa Heart Study who were 20 to 38 years of age. MEASUREMENTS: The authors performed advanced lipoprotein testing using vertical-spin density-gradient ultracentrifugation, traditional testing using enzymatic methods, and Friedewald formula estimation of low-density lipoprotein cholesterol levels. A certified reader blinded to lipoprotein results determined carotid intima-media thickness by B-mode ultrasonography. C-statistics from area under the receiver-operating characteristic curves (AUCs) derived from multivariable regression models were compared. RESULTS: Lipid values obtained with advanced lipoprotein testing did not predict carotid intima-media thickness better than traditionally measured lipid values in 236 participants for whom all data were available. A model using traditional lipoprotein measures (AUC, 0.754 [95% CI, 0.690 to 0.812]) did not differ significantly from a model using advanced lipoprotein measures (AUC, 0.779 [CI, 0.662 to 0.871]) for prediction of carotid intima-media thickness (P > 0.2). Subclass pattern of LDL, lipoprotein(a) cholesterol, intermediate-density lipoprotein cholesterol, high-density lipoprotein cholesterol subclasses, and very-low-density lipoprotein subclasses did not improve the performance of models for prediction of carotid intima-media thickness. LIMITATIONS: The study was cross-sectional, cardiac events were not determined, and only 1 method of advanced lipoprotein testing was used. CONCLUSIONS: Advanced lipoprotein testing using vertical-spin density-gradient ultracentrifugation did not improve prediction of carotid intima-media thickness in young adults and may not be useful for assessing cardiovascular risk in this population.

Adult↗

PubMed1976

Bovine secretory component. Isolation, molecular size and shape, composition, and NH2-terminal amino acid sequence.

Bovine free secretory component was purified from whey by salt precipitation, gel filtration, DEAE-cellulose and phosphocellulose chromatography, and immunoadsorption. It was obtained in immunologically pure form and in 56% yield. The Stokes radius of pure free secretory component was found to be 4.3 nm by gel filtration, and an (see article) of 4.1 S was determined by the ultracentrifuge. The molecular weight was 79,000 by sodium dodecyl sulfate gel electrophoresis and by sedimentation dquilibrium in the ultracentrifuge, using a v of 0.73 determined by ultracentrifugation in D2O and H2O. A minimal axial ratio of approximately 5 was calculated. Amino acid analysis of bovine free secretory component showed remarkable similarity to that of human, dog, and rabbit but carbohydrate analysis showed significant differences. In contrast to the human, bovine free secretory compoennt has 2 methionine residues/mol. The NH2-terminal sequence was found to be Lys-Ser-Pro-Ile-PPHE-Gly-Pro-Glu-Glu-Val-Asp-Ser-Val. This sequence is identical with that the human and dog. However, the poor immunological cross-reactivity between the dog, human, and bovine proteins suggests that significant structural differences will be found in other regions of the molecule.

Amino Acid Sequence↗

PubMed1975

Platelet contractile proteins: separation and characterization of the actin and myosin-like components.

Solution of thrombosthenin, the contractile protein complex isolated from pig platelets, have been studied by analytical ultracentrifugation and zone sedimentation in sucrose density gradients. Freshly prepared thrombosthenin in 0.6 M KCl shows a prominent peak in the ultracentrifuge with S degrees 20w about 5.5 and higher molecular weight aggregates (greater than 100S) sedimenting quickly to the bottom of the cell. Short term storage of high ionic strength solutions of thrombosthenin induces actomyosin-like gel formation and these gels dissociate with ATP and Mg2+ ions into two components of S degrees 20w 8.0 and S degrees 20w50. The supernatant, after actomyosin gel removal, contains only the S degrees 20w5.5 protein. From results of Ca2+ ATPase activity measurements and SDS polyacrylamide gel electrophoretic mobilities of dissociated thrombosthenin separated into fractions in sucrose density gradients, it is concluded that the S degrees20w5.5 protein species is the myosin-like protein of thrombosthenin. The S degrees 20w8.0 protein is not fibrinogen but also has myosin-like properties and is believed to be myosin dimer. Species of higher S values seen in the presence of ATP and Mg2+ in the analytical ultracentrifuge and located in the higher density zones of the sucrose gradients all gave in SDS polyacrylamide gel electrophoresis a single band of molecular weight 46-47,000 daltons. These subunit proteins appear to be derived from a range of polymeric variants of the F-actin-like protein of the contractile complex. All these higher density F-actin-like proteins readily form superprecipitates and display syneresis when combined with rabbit skeletal muscle myosin or platelet myosin. They are also all capable of conferring upon these two myosins a Mg2+ activated ATPase activity. It is suggested that in thrombosthenin solutions a myosin monomer-dimer equilibrium state exists which can be directionally influenced by a number of factors. The coexistence in the solution of F-actin and Mg2+ ATP, for example, increases the propensity of the myosin-like protein to form the higher molecular weight aggregate. Such aggregation may be the initiating mechanism for the intracellular organization of the thick filaments of the actomyosin complex, preparatory to a contractile event.

Actins↗

PubMed2005

Comparison of Golgi apparatus and endoplasmic reticulum proteins from livers of juvenile and aged rats using a novel technique for separation and enrichment of organelles.

The broad dynamic range of protein abundances, which can vary from about 10(6) for cells to 10(10) for tissues in complex proteomes, continues to challenge proteomics research. Proteome analysis, in particular organelle proteomics, using current approaches, requires extensive fractionation, separation, and enrichment. Over the years, organelle separation was achieved through the use of differential and density-gradient ultracentrifugation. However, the traditional fixed-volume process is a time-consuming and labor-intensive method, especially with large quantities of sample. Here, we present a novel tool for subcellular fractionation of biologically complex mixtures: continuous-flow ultracentrifugation of tissue homogenates to obtain both organelle separation and extensive organelle enrichment at the same time. In this study, rat liver tissues from two different age groups (3-8 wk and greater than 1 y old) were homogenized by blending. After removing nuclei, the resulting homogenates were further fractionated at the subcellular level by the use of sucrose gradient continuous-flow ultracentrifugation. Each organelle's enriched fractions were identified by Western blot analysis. To study the possible effects of aging on the endoplasmic reticulum and Golgi apparatus, we compared the organelle protein profiles of the two groups of rat liver tissues using two-dimensional gel electrophoresis, digitized imaging of two-dimensional gel electrophoresis, and mass spectrometry. Significant differences in the protein profiles of both organelles were observed between the two groups of rat tissues. The technique described here for fractionation and enrichment of organelles demonstrated a useful tool for proteomics research, including identification of low-abundance proteins and post-translational modifications.

Aging↗

PubMed2005

Proteomic changes in bovine heart mitochondria with age: using a novel technique for organelle separation and enrichment.

Separation and enrichment of organelles from complex biological mixtures are important for proteomic analysis. Two widely used current standard techniques to isolate individual organelles include differential and density-gradient centrifugation. Although these techniques have proven useful for processing small volumes of sample, multiple rounds of centrifugation are required when performing a large-scale purification. In this report, we have introduced a novel technique: continuous-flow ultracentrifugation using a sucrose gradient to separate, accumulate, and highly enrich bovine heart mitochondria in one step. To demonstrate the advantage of the technique, mitochondrial proteins from two different bovine hearts (3-8 mo and 18-30 mo old) were examined. For each age group, 100 g of bovine heart tissue were homogenized by a blending procedure. After removal of the nuclei, the entire remaining homogenate was loaded onto a proteomics continuous-flow ultracentrifuge to separate and enrich the organelles. Fractions were collected and mitochondria-enriched fractions were identified by Western blot analysis. To study the protein profile changes with aging in the mitochondrial proteome, the mitochondria-enriched fractions were applied to two-dimensional gel electrophoresis. The resulting two-dimensional PAGE gels were subsequently analyzed by image analysis software to identify proteins unique to each age group and proteins with at least twofold differences in protein expression. These proteins were then digested with trypsin and identified by mass spectrometer. Significant differences in the protein profiles of the two differently aged mitochondria preparations were found. The continuous-flow ultracentrifugation technique was demonstrated to be a powerful tool for separation and enrichment of organelles and their sub-types.

Aging↗

PubMed1976

Studies on human serum high density lipoproteins. Self-association of apolipoprotein A-I in aqueous solutions.

Human serum apolipoprotein A-I (apo-A-I), the major protein component of the human serum high density lipoproteins, was studied in aqueous solutions of differing ionic strength and pH by the techniques of sedimentation equilibrium ultracentrifugation and frontal analysis gel chromatography. The ultracentrifugal studies indicate the apo-A-I is a self-associating system that is dependent upon protein concentration, but relatively independent of the nature of the medium. The apparent weight average molecular weights obtained from solutions of initial apo-A-I concentration between 0.2 and 0.9 mg/ml were in the range of 3.0 to 16.7 x 10(4) (monomer molecular weight = 28,014). Of the several models of self-associated examined, that which gave the best theoretical fit was for the monomer-dimertetramer-octamer model. The self-association of apo-A-I in aqueous solutions was further documented by frontal analysis gel chromatography, which not only corroborated the ultracentrifugal results, but also indicated that the multiple species of apo-A-I in solution attain equilibrium rather rapidly. Besides having intrinsic importance, these results indicate that the solution properties of apo-A-I must be established before ligand binding studies are conducted and interpreted.

Apoproteins↗

PubMed1988

Quick method of determining lipoproteins, including those of intermediate density, in serum.

We describe an ultracentrifugation method for isolating the different lipoprotein classes relatively quickly. In this method the very-low-density lipoproteins are first separated by non-density-adjusted ultracentrifugation. The resulting infranatant material is then stained with Coomassie Brilliant Blue R-250 and ultracentrifuged in a density gradient. The intermediate-density lipoproteins (IDL), low-density lipoproteins, and high-density lipoproteins fractions are separated by aspiration from the top of the tube. This method can be used to separate, analyze, and quantify lipoproteins, including anomalous lipoproteins such as the IDL. The CVs for the present method never exceeded 15%.

Blood Proteins↗

PubMed1983

C3 convertase of the alternative complement pathway. Demonstration of an active, stable C3b, Bb (Ni) complex.

The purposes of this study were to demonstrate the C3 convertase complex, C3b, Bb (EC 3.4.21.47), of the alternative pathway of complement by ultracentrifugation and to determine whether the metal ion required for enzyme formation is present in the active enzyme complex. It has been shown previously that C3b,Bb formed with Ni2+ rather than Mg2+ exhibits enhanced stability. Using sucrose density gradient ultracentrifugation, an enzymatically active C3b,Bb(Ni) complex could be demonstrated which has a sedimentation coefficient of 10.7 S and which is stable in 10 mM EDTA. Upon formation of the enzyme with the radioisotope 63Ni2+, the ultracentrifugal distribution of the metal correlated with that of the enzyme complex. The molar ratio of Ni to C3b,Bb was 1:1. Displacement of Ni by Mg during formation of the enzyme indicated that both metals may bind to the same site in the enzyme. Binding of 63Ni to the catalytic site bearing fragment Bb was significantly stronger than its binding to C3b or to the zymogen, Factor B. It is proposed that there is one metal-binding site in the C3b,Bb enzyme which is not susceptible to chelation by EDTA and which is located in the Bb subunit.

Complement Activating Enzymes↗

PubMed1984

Five methods for determining low-density lipoprotein cholesterol compared.

We evaluated three precipitation methods for determination of low-density lipoprotein cholesterol in serum and an indirect method involving the Friedewald formula (Clin Chem 18: 499-502, 1972) by comparison with results by ultracentrifugation. The results of all methods for 83 sera, including 59 hyperlipidemic type IIA, IIB, and IV sera agreed very well, at least for concentrations of serum triglycerides below 8 mmol/L. The accuracy of the Friedewald formula was confirmed in 285 other sera, including 66 sera with triglycerides content between 4.52 and 8.0 mmol/L. For type III sera, the precipitation methods produced similar values to those obtained with the Friedewald formula, all being much higher than the ultracentrifugation values. Density-gradient ultracentrifugation showed that the very-low-density lipoprotein remnants in type III sera almost completely coprecipitated with the low-density lipoproteins. The precipitation methods are not only accurate but also very precise (CV less than 5%); they can therefore be used in clinical laboratories to measure atherogenic low-density lipoproteins plus the remnants of very-low-density lipoproteins. However, when serum triglycerides and high-density lipoprotein cholesterol also are determined, the Friedewald formula is a reliable alternative.

Centrifugation, Density Gradient↗

PubMed1984

A study of the small spherical high density lipoproteins of patients afflicted with familial lecithin: cholesterol acyltransferase deficiency.

We studied the effects of the lecithin:cholesterol acyltransferase reaction on the size and composition of the small spherical high density lipoproteins of patients afflicted with familial lecithin:cholesterol acyltransferase deficiency. We isolated these lipoproteins by preparative ultracentrifugation and rate zonal ultracentrifugation, determined their diameter by gradient gel electrophoresis, and then calculated their composition by relating measurements of their lipid and apolipoprotein content to particle volume. Our results revealed lipoprotein particles 6.0-6.2 nm in diameter that contained approximately 2 molecules of apolipoprotein A-I, 37-38 molecules of phospholipid, 3-9 molecules of unesterified cholesterol, 1-2 molecules of cholesteryl ester, and 1-2 molecules of triacylglycerol. Upon being incubated with lecithin:cholesterol acyltransferase and a source of additional unesterified cholesterol, these lipoproteins increased in content of total cholesterol and in particle size to form discrete lipoprotein products 6.6-8.6 nm in diameter. The increase in size occurred despite a net decrease in product unesterified cholesterol and phospholipid and though the net change in total lipid volume was small. Moreover, specific product lipoproteins, isolated by rate zonal ultracentrifugation, contained an increased amount of apolipoprotein A-I. These results seem best explained by a process involving lecithin:cholesterol acyltransferase-induced particle rearrangement reactions. The possibility that a similar process normally occurs in vivo deserves to be explored.

Electrophoresis, Polyacrylamide Gel↗

PubMed1981

Glycosphingolipid-high density lipoprotein-3 interactions. I. Transfer of glycosphingolipid from phosphatidylcholine vesicles to high density lipoprotein-3.

Single bilayer vesicles (d less than 1.02 g/ml) of 3H-glycosphingolipids and [14C]phosphatidylcholine in the molar ratio of 1:7 were prepared by ethanolic injection of the lipid mixture into buffer, concentrated, and incubated with human serum high density lipoprotein-3 (HDL3; d = .14 g/ml) at 37 degrees C. Equilibrium ultracentrifugation of the incubation mixtures on a 0-22% NaBr gradient revealed the presence of three discrete lipid-protein complexes of density 1.03, 1.06, and 1.12 g/ml (Peaks I, II, and III, respectively). Each peak was homogeneous upon reultracentrifugation and the protein and radioactivity eluted as a single peak upon Sepharose CL-6B chromatography. Compositional analysis showed peak I to contain 2.6% protein (apo-A-I peptide) and 4.3% cholesterol, peak II to contain 17.6% protein (apo-A-I peptide) and 6.3% cholesterol, and peak III to have a composition similar to HDL3. Electron microscopy of negatively stained samples confirmed the homogeneity of the peaks and the similarity between peak III and HDL3. Peak II particles were larger than HDL3; peak I particles resembled fused or aggregated vesicles which could be removed by ultracentrifugation; disc-shaped particles were not seen in any of the fractions. Direct incubation of HDL3 or human serum with 3H-glycosphingolipid dispersions did not yield a glycolipid . HDL3 complex as judged by density gradient ultracentrifugation and Sepharose CL-6B chromatography. However, incubation of 3H-glycolipid/phosphatidylcholine vesicles with serum did result in transfer of 3H-glycolipid to the HDL fraction. It was concluded that glycolipids incorporated into a lipid membrane structure can interact with, and become incorporated into, high density lipoprotein.

Gangliosidoses↗

PubMed1982

Changes in the distribution and composition of plasma high density lipoproteins after ingestion of fat.

Following ingestion of a fatty meal there is an increase in concentration of phospholipids and proteins in the plasma high density lipoproteins (HDL). To evaluate the resulting changes in HDL subclasses, the plasma HDL of six subjects were analyzed 4 to 8 h after ingestion of 100 ml of corn oil or 80 ml of corn oil with four eggs. Isopycnic density gradient ultracentrifugation of fasting plasma showed two broad components of HDL: a major peak of density (d) 1.11 to 1.17 g/ml (HDL3) and a smaller peak of d 1.07 to 1.11 g/ml (HDL2). Following ingestion of either type of fatty meal, there was an increase in lipoprotein mass in both peaks of HDL and their centers of mass were shifted to lower density (1.140 leads to 1.120 to 1.130 g/ml; 1.095 leads to 1.090 g/ml). Calculation of changes in HDL concentration (lipemic minus fasting) showed that the alterations in density gradient profile were due to a major increase in lipoproteins of d 1.102 to 1.137 g/ml, a smaller increase in a separate lipoprotein peak of 1.080 to 1.102 g/ml, and a small decrease in lipoproteins of d 1.137 to 1.165 g/ml. Redistribution of HDL mass into larger, less dense lipoproteins was also demonstrated by agarose gel chromatography or by minimal spin density gradient ultracentrifugation in a vertical rotor. The increase in mass of 1.080 to 1.102 lipoproteins was largely due to increased concentrations of phospholipid, cholesterol ester, and apoA-I, while the increase in 1.102 to 1.137 lipoproteins was due to increased concentrations of apoA-I, apoA-II, phospholipids, cholesterol, and cholesterol esters. Analytical ultracentrifugation of representative samples within these density intervals showed lipoprotein species with molecular weights and sedimentation coefficients, respectively, of 378,000, 5.8 (d 1.080 to 1.095); 248,000, 3.5 (d 1.110 to 1.120); and 173,000, 1.6 (d 1.135 to 1.150). Polyacrylamide gradient gel electrophoresis showed that the 1.080 to 1.102 lipoproteins contained a single lipoprotein band of diameter approximately 10.7 nm; the 1.102 to 1.137 lipoproteins contained a single band which varied in size fro 10.0 to 9.2 nm: and the 1.137 to 1.165 lipoproteins contained three species of diameters approximately 9.2, 8.8, and 8.2 nm. Within density intervals, the molecular weights, sedimentation coefficients, and diameters of the different lipoproteins were similar in fasting and lipemic plasma. Calculation of average molecular compositions shows that the major incremental HDL of d approximately 1.12 g/ml could be derived by addition of lipids to the largest species of fasting HDL3. Within density intervals, the particle contents of apoA-I and apoA-II were unchanged during lipemia, suggesting that apoprotein transfer causes interconversion of existing HDL species or formation of new particles with the same content of apoA-I and apoA-II as existing species.

Adult↗

PubMed1978

Composition of proteins of mesenteric lymph chylomicrons in the rat and alterations produced upon exposure of chylomicrons to blood serum and serum proteins.

Protein composition was determined in mesenteric lymph chylomicrons from fat-fed rats. Among the proteins of intermediate molecular weight, apoproteins A-I and the arginine-rich apoprotein accounted for 31% and 4% of the total protein mass, respectively. Apoprotein B and apoprotein A-IV each accounted for about 10% and proteins of low molecular weight (C apoproteins and apoprotein A-II) accounted for most of the remainder. Apoprotein A-I also accounted for more than 30% of the protein mass of mesenteric lymph lipoproteins of density less than 1.006 g/ml ("small chylomicrons") obtained from rats fed glucose. Aproprotein A-I was partially dissociated from chylomicrons during brief ultracentrifugation. Both the arginine-rich apoprotein and the C apoproteins in rat blood serum were transferred to lymph chylomicrons from fat-fed rats during incubation in vitro. Content of arginine-rich apoprotein, determined immunochemically, increased six-fold when chylomicrons were diluted to a final concentration of 500 mg/dl in blood serum. Upon incubation of chylomicrons in equivalent volumes of ultracentrifugal fractions of serum, the increase of the arginine-rich apoprotein was: very low density lipoproteins, 1.5-fold; high density lipoproteins, 1.8-fold; density fraction greater than 1.006 g/ml, 5.0-fold; density fraction greater than 1.21 g/ml, 11-fold. Content of apoprotein A-I, also determined immunochemically, was not altered appreciably by exposure to serum or its ultracentrifugal fractions, whereas content of C apoproteins, estimated from intensity of staining of the low molecular weight protein component in polyacrylamide gel electropherograms, increased in all cases except for the density fraction greater than 1.21 g/ml. The fractional content of apoprotein A-I in the protein of chylomicrons fell after incubation, whereas that of the arginine-rich apoprotein remained constant or rose substantially. The fractional content of apoprotein A-IV in chylomicron-protein tended to follow that of apoprotein A-I, as judged from polyacrylamide gel electropherograms. Transfer of the arginine-rich and C apoproteins to chylomicrons from blood serum was directly related to the volume of serum in which the chylomicrons were diluted and occurred rapidly at room temperature or at 4 degrees C.

Animals↗

PubMed1982

Interactions of bacterial lipopolysaccharide with acute-phase rabbit serum and isolation of two forms of rabbit serum amyloid A.

Mixing lipopolysaccharide (LPS, Salmonella minnesota R595) with acute-phase rabbit serum (APRS) results in the formation of two types of complexes. The two complexes are separable from each other and from LPS by CsCl density gradient ultracentrifugation. LPS alone had density 1.38 g/cm3, complex 1.3 had density 1.3 +/- 0.02 g/cm3, and complex 1.2 had density less than 1.20 g/cm3. At 1 to 10 micrograms LPS/ml APRS, up to 23 micrograms of LPS could be found in complex 1.3, with the remainder of the LPS in complex 1.2. The capacity of complex 1.2 for LPS was 500 to 600 micrograms LPS/ml APRS. The ability of rabbit serum to form complex 1.3 rose to a maximum in 24 hr post-acute-phase induction. The two complexes were purified by equilibrium density gradient ultracentrifugation and immunoaffinity chromatography using antibodies to LPS and subjected to SDS-PAGE. Complex 1.3 had major peptides after reduction of 91, 64, 61 and 50 kilodaltons. The 61-kilodalton peptide is probably rabbit serum albumin; the others are unidentified. Complex 1.2 had major peptides after reduction whose mobility corresponded to apolipoprotein A-I and serum amyloid A. Complex 1.2 thus seems to be analogous to the LPS-high-density-lipoprotein complexes that form in normal serum except that the latter complexes do not contain serum amyloid A. Rabbit serum amyloid A (SAA) was purified by CsCl equilibrium density gradient ultracentrifugation, gel filtration, and ion-exchange chromatography into two forms, SAA-1 and SAA-2. The two forms of SAA have very similar amino acid compositions and m.w. (SAA-1, 11,526 daltons; SAA-2, 11,469 daltons). They differ primarily in their isoelectric points, 6.57 and 6.27 for SAA-1 and SAA-2, respectively. The complexes 1.2 from several individual rabbits after acute-phase induction were studied by isoelectric focusing. Seven of 10 rabbits showed both forms of SAA; three showed a single form. Two rabbits showing SAA-1 on the first acute-phase induction were reinduced; one rabbit again gave SAA-1 and the other gave SAA-2. These results suggest that SAA and perhaps other acute-phase reactants may modulate the biologic activity of LPS.

Acute Disease↗