Separation and analysis of lipoproteins by gel filtration

In an attempt to provide immunological tools for subfractionation of high-density lipoproteins (HDL), monoclonal antibodies were raised against HDL complexes. Two clones identified a peptide, provisionally named K-45 (PI 4.5-4.9; molecular mass 45 kDa, range 42-48 kDa), whose plasma distribution and lipoprotein association were fully characterised. Gel filtration localised the peptide to the HDL region of human plasma where it co-eluted with apolipoprotein (apo) A-I, the structural protein of HDL. Complementary studies employing immunoabsorption with anti-(apo A-I) antibodies removed 90 % of K-45 from plasma: conversely, anti-(apo A-11) antibodies eliminated only 10 % of K-45. Immunoaffinity chromatography on an anti-(K-45) column revealed that the peptide was present in a distinct HDL subspecies containing three major proteins: K-45, apo A-I and clusterin or apo J. The lipoprotein nature of the bound fraction was indicated by electron microscopy (diameter 9.6 ? 3.3 nm) and quantification of lipids, the latter showing an unusually high triacyglycerol concentration. Plasma concentrations of K-45 were positively correlated with apo A-I and HDL-cholesterol and negatively correlated with apo B and total cholesterol. Thus, the peptide appears to be linked, directly or indirectly, to processes which give rise to an anti-atherogenic lipid profile. After completion of the present studies, an N-terminal sequence identical to that of K-45 was reported in recently isolated cDNA clones. These clones encode paraoxonase.Lipoprotein complexes are the principal transport vehicles for plasma lipids. As such, they are the focus of particular attention which derives from the designation of blood lipids as primary cardiovascular risk factors [l, 21. The rationale behind these studies is that a dysfunctional lipid transport system will be a major cause of the dyslipidaemias associated with premature cardiovascular disease [3]. In this context, an obvious pre-requisite is a comprehensive understanding of the normal functioning of the lipoprotein metabolic system ; unfortunately, this is not currently the case. It is due, in part, to the highly dynamic nature of this metabolic process, rendered even more intricate by extensive interactions between the major subclasses, very-low-density (VLDL), low-density (LDL) and high-density (HDL) lipoproteins [4]. The latter are of particular interest as they afford a measure of protection against cardiovascular disease [5, 61. Yet many aspects of HDL metabolism are poorly understood. Neither the origins nor the sites of catabolism of this lipoprotein species have been convincingly demonstrated. Additionally, the mechanisms by which HDL assure their postuCorrespondence to R.

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“…and NaN, (0.01 % mass/vol.) [27]. Fractions of 2.0ml were collected and analysed for lipids and protein.…”

Section: Materials and Methods Preparation Of Lipoproteinsmentioning

confidence: 99%

Identification of a distinct human high‐density lipoprotein subspecies defined by a lipoprotein‐associated protein, K‐45

Blatter

James

Messmer

et al. 1993

European Journal of Biochemistry

322

189

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“…Interaction Selectivity of HuPON1 with Amphiphilic Aggregates-In serum, HuPON1 was exclusively found associated with HDL-type complexes, which differ in size and composition from the much larger low density and very low density lipoproteins of 20-nm and 30 -80-nm diameter, respectively (45). As hypothesized by Oda et al (46), since the HuPON1-HDL lipoparticles characterized in plasma were mainly found associated with apoA-I (9, 17) and HDL-like complexes containing apoA-I were found to be necessary for optimal release and stabilization of HuPON1 (10), the association specificity of HuPON1 with HDL might rely on transient interaction of HuPON1 with apoA-I.…”

Section: Structural and Functional Similarities Between Hdl-and Determentioning

confidence: 99%

Oligomeric States of the Detergent-solubilized Human Serum Paraoxonase (PON1)

Josse¹,

Ebel

Stroebel³

et al. 2002

Journal of Biological Chemistry

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Human plasma paraoxonase (HuPON1) is a high density lipoprotein (HDL)-bound enzyme exhibiting antiatherogenic properties. The molecular basis for the binding specificity of HuPON1 to HDL has not been established. Isolation of HuPON1 from HDL requires the use of detergents. We have determined the activity, dispersity, and oligomeric states of HuPON1 in solutions containing mild detergents using nondenaturing electrophoresis, size exclusion chromatography, and crosslinking. HuPON1 was active whatever its oligomeric state. In nonmicellar solutions, HuPON1 was polydisperse. In contrast, HuPON1 exhibited apparent homogeneity in micellar solutions, except with CHAPS. The enzyme apparent hydrodynamic radius varied with the type of detergent and protein concentration. In C 12 E 8 micellar solutions, from sedimentation velocity, equilibrium analytical ultracentrifugation, and radioactive detergent binding, HuPON1 was described as monomers and dimers in equilibrium. A decrease of the detergent concentration shifted this equilibrium toward the formation of dimers. About 100 detergent molecules were associated per monomer and dimer. The assembly of amphiphilic molecules, phospholipids in vivo, in sufficiently large aggregates could be a prerequisite for anchoring of HuPON1 and then allowing stabilization of the enzyme activity. Changes of HDL size and shape could strongly affect the binding affinity and stability of HuPON1 and result in reduced antioxidative capacity of the lipoprotein.

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“…This method has been extensively validated by Zilversmit and Shea, 27 and in control experiments we found that it gave results similar to those obtained by the method of Lowry et al 28 LDL protein was converted to total LDL mass using molecular weights of apoB and LDL of 0.5ϫ10 6 g/mol and 3ϫ10 6 g/mol, respectively. 29,30 Lipoprotein Labeling Lp(a) [4.5 to 7.4 mg total Lp(a) mass] or LDL (5 mg protein, 30 mg total LDL mass) was labeled with 150 to 185 MBq *I, Amersham, Birkerød, Denmark) using ICl. 23,31,32 TC (50 nmol) was labeled with 150 to 185 MBq *I using Iodogen (Pierce Chemical Company, Aarhus, Denmark).…”

Section: Isolation Of Human Lp(a) and Ldlmentioning

confidence: 99%

Increased Degradation of Lipoprotein(a) in Atherosclerotic Compared With Nonlesioned Aortic Intima–Inner Media of Rabbits

Nielsen

Juul

Nordestgaard

1998

ATVB

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Abstract-To investigate a potential role of lipoprotein(a) [Lp(a)] in foam cell formation, we have measured the degradation rates of Lp(a) and LDL in the rabbit aorta in vivo. Lp(a) (or LDL) was labeled with both 131 I-TC and 125 I and injected into 17 rabbits with extensive aortic atherosclerosis and into 16 rabbits without atherosclerosis. As the protein moiety of the doubly labeled lipoproteins is degraded, 131 I-TC is trapped in the cell, whereas 125 I diffuses out of the cell. Twenty-four hours after injection, 12 samples of the aorta and biopsies from 9 other tissues were removed. The degradation rate of Lp(a) (percent of plasma pool per gram tissue per day) was less than that of LDL in the adrenals and in the intestine. In contrast, degradation rates of Lp(a) and LDL were similar in liver, spleen, kidney, heart, lung, skeletal muscle, and adipose tissue. In nonlesioned aortic intima-inner media, the degradation rate of Lp(a) was 39% of that of LDL (t test: P Ͻ.05 in aortic arch and thoracic aorta), whereas the degradation rates of Lp(a) and LDL were similar in atherosclerotic aortic intima-inner media. Lp(a) degradation rates were markedly increased in atherosclerotic compared with nonlesioned aortic intima-inner media: 28.2Ϯ9.2ϫ10 Ϫ7 % and 5.0Ϯ0.6ϫ10 Ϫ7 % of the plasma pool per gram tissue per day in the intima-inner media of the proximal segment of atherosclerotic and nonlesioned aorta, respectively (t test: P Ͻ.01). These results suggest that the metabolism of Lp (a) phospholipids, triglycerides, and apoB. 1 In epidemiological studies, high plasma levels of Lp(a) have been associated with an increased risk of cardiovascular disease. 2 The mechanism by which Lp(a) may promote atherothrombotic disease is unclear, but several possibilities are suggested by the structure of Lp(a). Lp(a) contains apo(a), which is attached to apoB through a disulfide bridge. 3 Apo(a) has a high degree of structural similarity to plasminogen, 4 and Lp(a) has been shown to inhibit plasmin formation in vitro. 5 As a result of this effect, Lp(a) inhibits fibrinolysis 5 and accelerates growth of smooth muscle cells 6 in vitro. Although these effects could promote the development of atherothrombotic disease, the significance needs to be determined in vivo.Because Lp(a) contains cholesterol and cholesterol esters, uptake and degradation of Lp(a) by monocyte-macrophages in the arterial intima could promote foam cell formation, the pathologic hallmark of early atherosclerosis. 7 In vitro evidence suggests that Lp(a) may be taken up and degraded by macrophage-derived foam cells via a cellular receptor distinct from the LDL receptor, scavenger receptors, the LDL receptor-related protein, or plasminogen receptors. 8 -10 Other studies have shown that Lp(a) can be degraded by monocytemacrophages via the LDL receptor and "nonspecific" pathways. [11][12][13][14][15][16] Complex formation by Lp(a) with glycosaminoglycans or modification of Lp(a) with malondialdehyde results in a markedly enhanced uptake and degradation of Lp(a) by monocyte...

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Separation and analysis of lipoproteins by gel filtration

Cited by 71 publications

References 15 publications

Identification of a distinct human high‐density lipoprotein subspecies defined by a lipoprotein‐associated protein, K‐45

Identification of a distinct human high‐density lipoprotein subspecies defined by a lipoprotein‐associated protein, K‐45

Oligomeric States of the Detergent-solubilized Human Serum Paraoxonase (PON1)

Increased Degradation of Lipoprotein(a) in Atherosclerotic Compared With Nonlesioned Aortic Intima–Inner Media of Rabbits

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