scholarly journals Plasma lipoprotein and apolipoprotein distribution as a function of density in the rainbow trout (Salmo gairdneri)

1987 ◽  
Vol 246 (2) ◽  
pp. 425-429 ◽  
Author(s):  
P J Babin
Keyword(s):  
Plasma Proteins ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
Single Type ◽  
Salmo Gairdneri ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Intermediate Density

I have previously described [Babin (1987) J. Biol. Chem. 262, 4290-4296] the apolipoprotein composition of the major classes of trout plasma lipoproteins. The present work describes the use of an isopycnic density gradient centrifugation procedure and sequential flotation ultracentrifugation to show: (1) the presence of intermediate density lipoproteins (IDL) in the plasma, between 1.015 and 1.040 g/ml; (2) the existence of a single type of Mr 240,000 apoB-like in the low density lipoproteins (LDL, 1.040 less than p less than 1.085 g/ml); (3) the presence of apoA-I-like (Mr 25,000) in the densest LDL; (4) the adequacy of 1.085 g/ml as a cutoff between the LDL and high density lipoproteins (HDL); (5) the accumulation of Mr 55,000 and 76,000 apolipoproteins and apoA-like apolipoproteins in the 1.21 g/ml infranatant. The fractionation of trout lipoprotein spectrum thus furnishes the distribution of the different lipoprotein classes and leads to the description of the constituent apolipoproteins, which account for about 36% of circulating plasma proteins in this species.

scholarly journals Consequences of Interaction of a Lipophilic Endotoxin Antagonist with Plasma Lipoproteins

2000 ◽  
Vol 44 (3) ◽  
pp. 504-510 ◽  
Author(s):  
Jeffrey R. Rose ◽  
Maureen A. Mullarkey ◽  
William J. Christ ◽  
Lynn D. Hawkins ◽  
Melvyn Lynn ◽  
...  
Keyword(s):  
Human Blood ◽  
Gradient Centrifugation ◽  
Low Density Lipoproteins ◽  
Drug Binding ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Cholesterol Concentration ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Plasma Fraction

ABSTRACT E5531, a novel synthetic lipid A analogue, antagonizes the toxic effects of lipopolysaccharide, making it a potential intravenously administered therapeutic agent for the treatment of sepsis. This report describes the distribution of E5531 in human blood and its activity when it is associated with different lipoprotein subclasses. After in vitro incubation of [14C]E5531 with blood, the great majority (>92%) of material was found in the plasma fraction. Analysis by size-exclusion and affinity chromatographies and density gradient centrifugation indicates that [14C]E5531 binds to lipoproteins, primarily high-density lipoproteins (HDLs), with distribution into low-density lipoproteins (LDLs) and very low density lipoproteins (VLDLs) being dependent on the plasma LDL or VLDL cholesterol concentration. Similar results were also seen in a limited study of [14C]E5531 administration to human volunteers. The potency of E5531 in freshly drawn human blood directly correlates to increasing LDL cholesterol levels. Finally, preincubation of E5531 with plasma or purified lipoproteins indicated that binding to HDL resulted in a time-dependent loss of drug activity. This loss in activity was not observed with drug binding to LDLs or to VLDLs or chylomicrons. Taken together, these results indicate that E5531 binds to plasma lipoproteins, making its long-term antagonistic potency dependent on the plasma lipoprotein composition.

Abstract 437: Optimizing a Prokaryotic Cell Free Expression System for the Generation of synthetic ApoB-Containing Lipoprotein Particles

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Humra Athar ◽  
Zhenghui G Jiang ◽  
Christopher J McKnight
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Expression System ◽  
Low Density Lipoproteins ◽  
Free Expression ◽  
Low Density ◽  
Prokaryotic Cell ◽  
Lipoprotein Particles ◽  
Cell Free Expression System

High serum levels of low density lipoproteins (LDL) is associated with increased risk of atherosclerosis. Apolipoprotein B (ApoB) is required for the assembly and secretion of chylomicrons and very low density lipoproteins (VLDL), the precursor of low density lipoproteins (LDL). Despite its clinical significance, the mechanism of the assembly of these ApoB containing lipoproteins is poorly understood. The assembly process is an interplay of several key components including but not limited to nascent ApoB, lipids, ER resident chaperones and importantly, microsomal triglyceride transfer protein (MTP). In the current study, we are trying to understand several unanswered questions in the mechanism of the lipoprotein assembly. We have used a novel prokaryotic cell-free expression system and lipids mimicking the ER membrane to produce particles that represent the early dense initiation particles formed in the ER. After optimizing several different conditions, we were able to make “synthetic” lipoproteins by cotranslational expression of constructs from the first 22% of ApoB tagged with a 6-histidine tag at the C-terminus (ApoB 22-His) with small unilamellar phosphatidylcholine (PC) vesicles and phosphatidylcholine:triolein (PC:TO) emulsions. After cotranslational interaction with lipids, these constructs migrate to a lower density in potassium bromide (KBr) density gradient centrifugation. Here we report a new ApoB 22 construct with a FLAG tag at the N-terminus in addition to the C-terminal His tag. The construct makes significant amount of soluble protein that is soluble in the cell free reaction. The two N- and C-terminal tags allow us to purify full length construct from any truncation products. In addition, the dual-tag approach will allow us to purify the synthetic lipoproteins directly from the cell free system, and thereby avoid the requirement for KBr density gradient centrifugation. This new strategy will provide far more efficient generation and purification of synthetic ApoB containing lipoprotein particles.

scholarly journals IMMUNOCHEMICAL STUDIES ON HUMAN PLASMA LIPOPROTEINS

1957 ◽  
Vol 105 (1) ◽  
pp. 49-67 ◽  
Author(s):  
Frederick Aladjem ◽  
Miriam Lieberman ◽  
John W. Gofman
Keyword(s):  
Human Plasma ◽  
Plasma Protein ◽  
Protein Fraction ◽  
Low Density Lipoproteins ◽  
High Density ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Plasma Protein Fraction ◽  
Free Plasma

Low density human plasma lipoproteins Sf 17+, Sf 13, and Sf 6, high density lipoproteins 2 and 3, and a lipoprotein-free plasma protein fraction were isolated from human plasma by ultracentrifugal methods. It was found that human plasma lipoproteins are immunochemically distinct from the non-lipoprotein containing plasma protein fraction. Lipoprotein fractions of a given hydrated density, isolated from different individuals, were found to be immunochemically indistinguishable by qualitative absorption tests. Qualitative antigenic differences were shown to exist between low density lipoproteins and high density lipoproteins. Quantitative precipitin reactions showed that low density lipoproteins Sf 6 and Sf 13 were immunochemically very similar. However, they differed with respect to the amount of antigen nitrogen required for maximum precipitation. Agar diffusion analyses were performed; the results suggest heterogeneity of lipoproteins by this criterion.

scholarly journals Characterization of plasma lipoproteins separated and purified by agarose-column chromatography

1974 ◽  
Vol 139 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Lawrence L. Rudel ◽  
Jason A. Lee ◽  
Manford D. Morris ◽  
James M. Felts
Keyword(s):  
Human Plasma ◽  
Column Chromatography ◽  
Low Density Lipoproteins ◽  
High Density ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Simple Method ◽  
Lipoprotein Class

1. A simple method for isolation of individual human plasma lipoprotein classes is presented. In this technique, lipoproteins are removed from plasma at d1.225 by ultracentrifugation, after which they are separated and purified by agarose-column chromatography. 2. Three major classes are obtained after agarose-column chromatography. Separation between classes is excellent; more than 95% of the lipoproteins eluted from the column are recovered in the form of a purified lipoprotein class. 3. Each lipoprotein class was characterized immunologically, chemically, electrophoretically and by electron microscopy. A comparison of the properties of the column-isolated lipoproteins was made with very-low-density lipoproteins, low-density lipoproteins, and high-density lipoproteins separated by sequential ultracentrifugation at densities of 1.006, 1.063 and 1.21 respectively. 4. By each criterion, peak-I lipoproteins from the agarose column are the same as very-low-density lipoproteins, peak-II lipoproteins are the same as low-density lipoproteins, and peak-III lipoproteins are the same as high-density lipoproteins. Thus the lipoprotein classes isolated by both methods are similar if not identical. 5. The agarose-column separation technique offers the advantage of a two- to three-fold saving in time. In addition, the column-elution pattern serves as a recording of the size distribution of lipoproteins in plasma. 6. The most complete characterization is reported for human plasma lipoproteins. The results with rhesus-monkey and rabbit lipoproteins were identical.

scholarly journals Membrane receptors for very low density lipoprotein (VLDL) inhibitor of lymphocyte proliferation

Blood ◽  
1981 ◽  
Vol 57 (6) ◽  
pp. 1055-1064 ◽  
Author(s):  
PI Yi ◽  
G Beck ◽  
S Zucker
Keyword(s):  
Dna Synthesis ◽  
Lymphocyte Proliferation ◽  
Human Lymphocytes ◽  
Low Density Lipoproteins ◽  
Membrane Receptors ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Scatchard Analysis ◽  
Low Density ◽  
Very Low Density Lipoproteins

Abstract Physiologic concentrations of human plasma very low density lipoproteins inhibit the DNA synthesis of lymphocytes stimulated by allogeneic cells or lectins. In this report we have compared the effects of isolated lipoproteins [very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)] and lipoprotein-depleted plasma (LDP) on DNA synthesis by phytohemagglutinin-stimulated human lymphocytes. The relative potency for the inhibition of lymphocyte proliferation was VLDL greater than LDL greater than HDL greater than LDP. Fifty percent inhibition of DNA synthesis was observed at a VLDL protein concentration of 1.5--2.0 microgram/ml. We have further demonstrated the presence of specific receptors for VLDL on human lymphocytes. Native VLDL was more effective than LDL in competing for 125I-VLDL binding sites. Subsequent to binding to lymphocytes, 125I-VLDL was internalized and degraded to acid- soluble products. Based on a Scatchard analysis of VLDL binding at 4 degrees C, the number of VLDL receptors per lymphocyte was estimated at 28,000 +/- 1300. Based on an estimated mean binding affinity for the VLDL receptor complex at half saturation of approximately 8.8 X 10(7) liter/mole, it is estimated that 91% of lymphocyte VLDL receptors are occupied at physiologic VLDL concentrations in blood. Although the immune regulatory role of plasma lipoproteins is uncertain, we suggest tha VLDL and LDL-In may maintain circulating blood lymphocytes in a nonproliferative state via their respective cell receptor mechanisms.

scholarly journals STUDIES ON THE IMMUNOCHEMISTRY OF HUMAN LOW DENSITY LIPOPROTEINS UTILIZING AN HEMAGGLUTINATION TECHNIQUE

1959 ◽  
Vol 110 (1) ◽  
pp. 113-122 ◽  
Author(s):  
William W. Briner ◽  
Jackson W. Riddle ◽  
David G. Cornwell
Keyword(s):  
Serum Proteins ◽  
Gradient Centrifugation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Normal Subjects ◽  
Low Density Lipoproteins ◽  
Lipoprotein Fraction ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Chicken Erythrocytes

Hemagglutination is a specific and sensitive technique for investigating the purity of lipoproteins and the immunologic relationships between low density lipoprotein fractions. The Sf 10–400 and Sf 3–9 lipoprotein fractions, isolated from human serum by dextran sulfate-density gradient centrifugation procedure and repurified by centrifugation appeared to contain only lipoprotein antigens since these fractions did not stimulate the production of antibodies against other serum proteins. Cross-absorption experiments with lipoproteins carried on "tanned" cells demonstrated that the Sf 3–9 lipoprotein fraction contains all the antigenic components of the Sf 10–400 lipoprotein fraction together with additional antigenic components not found in the Sf 10–400 lipoprotein fraction. Thus Sf 3–9 and Sf 10–400 lipoprotein fractions are immunologically similar but not identical. Low density lipoproteins contain no antigens in common with the high density lipoproteins. An Sf 3–9 antiserum can be used to detect both Sf 3–9 and Sf 10–400 antigens. The Sf 3–9 lipoprotein fraction used as an antigen will detect antibodies against both Sf 3–9 and Sf 10–400 lipoprotein fractions. The Sf 3–9 and Sf 10–400 antisera did not contain immune antibodies against erythrocytes of the different blood groups or against sheep, guinea pig, dog, calf, pig, horse, and chicken erythrocytes. Normal subjects and subjects with recent myocardial infarctions had no circulating autoantibodies against the Sf 3–9 and Sf 10–400 lipoprotein fractions.

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

1988 ◽  
Vol 34 (9) ◽  
pp. 1753-1757 ◽  
Author(s):  
N Fontanals-Ferrer ◽  
J Serrat-Serrat ◽  
A Sorribas-Vivas ◽  
C Gonzalez-Garcia ◽  
F Gonzalez-Sastre ◽  
...  
Keyword(s):  
Density Gradient ◽  
Present Method ◽  
Low Density Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Quick Method ◽  
Coomassie Brilliant Blue ◽  
Coomassie Brilliant ◽  
Intermediate Density

Abstract 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%.

scholarly journals Inhibition of lymphocyte proliferation stimulated by lectins and allogeneic cells by normal plasma lipoproteins.

1977 ◽  
Vol 146 (6) ◽  
pp. 1791-1803 ◽  
Author(s):  
J H Morse ◽  
L D Witte ◽  
D S Goodman
Keyword(s):  
Human Plasma ◽  
Rank Order ◽  
Human Lymphocytes ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Normal Plasma

Lipoproteins, isolated by sequential flotation at densities 1.006, 1.019, 1.063, and 1.21, were examined for their ability to inhibit human lymphocytes stimulated by allogeneic cells and by lectins (phytohemagglutinin-P and concanavalin A). All the classes of normal plasma lipoproteins inhibited lymphoproliferation when peripheral blood lymphocytes were cultured in autologous, heterologous, or lipoprotein-deficient plasma (d greater than 1.21). The rank order of inhibitory potency was intermediate density lipoprotein (IDL) greater than very low density lipoproteins (VLDL) greater than low density lipoproteins (LDL) greater than high density lipoproteins (HDL), regardless of the mode of stimulation. The concentrations of IDL, VLDL, and LDL required for complete inhibition of stimulated lymphoproliferation were considerably below the levels of each of these lipoproteins normally found in human plasma. In addition, the concentration of HDL required for 50-90% inhibition was in the range of HDL levels normally found in human plasma. Moreover, at relatively higher concentrations, lipoproteins suppressed the incorporation of [3H]thymidine into DNA below the levels seen with reseting, unstimulated lymphocytes. The results suggest that circulating lymphocytes may normally be highly suppressed by the combined effects of all the endogenous lipoproteins and that the lipoproteins may play important roles in vivo in modulating lymphocyte functions and responses.

HUMAN PLASMA PAF-ACETYLHYDROLASE, NORMALLY PRESENT IN LOW DENSITY LIPOPROTEINS, IS ASSOCIATED WITH HIGH DENSITY LIPOPROTEINS IN A PATIENT WITH LDL DEFICIENCY

1987 ◽  
Author(s):  
I E Surya ◽  
J W N Akkerman
Keyword(s):  
Enzyme Activity ◽  
Gradient Centrifugation ◽  
Platelet Activating Factor ◽  
Autosomal Recessive Disorder ◽  
Low Density Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Apo B ◽  
Inflammatory Reactions ◽  
Paf Acetylhydrolase

Platelet Activating Factor (l-0-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; PAF) plays an important role in allergic and inflammatory reactions and activates platelets in the nanomolar range. One of the main factors that controls PAF activity in blood is an enzyme that hydrolyzes the acetyl-chain thereby converting PAF to biologically inactive lyso-PAF. The enzyme is acid labile and normally associated with apo B-containing low density lipoproteins (LDL, density 1,006-1,063 g/ml).We investigated whether a deficiency in LDL would affect the enzyme activity. PAF-inactivating activity was measured in plasma from a patient with abetalipoproteinemia, a rare autosomal recessive disorder, characterized by the absence of apo B and apo B-containing lipoproteins (chylomicrons, VLDL and LDL). Plasma triglyceride was 0,2 mmol/1 (normal 1,40-2,20 mmol/1) and cholesterol 1,3 mmol/1 (normal 5,60-7,70 mmol/1). Separation of lipoproteins by density gradient centrifugation revealed a slightly decreased HDL content whereas VLDL and LDL were below the detection limit (0,20 mmol/1; based on cholesterol content).Despite the absence of LDL, PAF-inactivating activity in plasma of the patient (measured by (1) the decrease in aggregation inducing activity of PAF after incubation, (ii) the conversion of 3H-acyl-PAF to lysa PAF, separated on TLC, (iii) the liberation of 3H-label from 3H-acetyl PAF) was present and even slightly higher than in normal plasma (hydrolysis of 3 3H-PAF after 20 minutes incubation was 78 ± 4% and 65 ± 6% in patient and normals, respectively, n = 4). Subfractionation revealed that the enzyme activity was present in fractions with densities of 1,065-1,214 g/ml, which are typical for HDL.These results indicate that PAF-acetylhydrolase, although normally present in LDL, binds to HDL in a patient with extreme LDL-deficiency.Supported by the Dutch Heart Foundation (grant 85082)

Diagnosis of Type III Hyperlipoproteinemia by Chromatography of Plasma Lipoproteins on Columns Containing Agarose

1975 ◽  
Vol 21 (13) ◽  
pp. 1887-1891 ◽  
Author(s):  
James Shepherd ◽  
Christopher J Packard ◽  
Frances J Dryburgh ◽  
Jane L H C Third
Keyword(s):  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Type Iii ◽  
Elution Pattern ◽  
Type Iii Hyperlipoproteinemia ◽  
Primary Type ◽  
Relative Deficiency

Abstract Agarose column chromatography has been used to separate plasma lipoproteins into very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). Applied to the diagnosis of primary type III hyperlipoproteinemia, the procedure is capable of demonstrating three characteristic and specific changes from normality in the elution pattern of lipoproteins from patients with this condition. In the type III profile there is (a) incomplete separation of VLDL from putative LDL material, (b) early elution of the type III LDL with respect to a normal LDL marker, and (c) relative deficiency of type III LDL with elution characteristics of normal LDL. We advocate the use of this method in the diagnosis of type III hyperlipoproteinemia.

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