Isolation and Characterization of Low Density Lipoproteins

Lee, Diana M.

doi:10.1007/978-1-4684-2250-4_1

Cited by 14 publications

(12 citation statements)

References 155 publications

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“…Under the assumption that Lp(a) and LDL bind to the same type of receptor, the differences in affinity and binding capacity could be due to the larger molecular weight of the Lp(a) particles, which is approximately twice as high as LDL (11,33). On the other hand, the specific Lp(a) antigen could possibly mask some of the apolipoprotein B recognition sites leading to altered steric effects of binding.…”

Section: Discussionmentioning

confidence: 99%

“…At day 5, the monolayers were washed with phosphate-buffered saline, after which Specific binding of '2I-Lp(a) or 1251-LDL was defined as total binding minus (nonspecific) binding seen in the presence of either unlabeled Lp(a) or LDL, at 500-Ag/ml concentrations. Binding activity is expressed as moles of radioligand bound to cells per culture dish, assuming a molecular weight of 5.6 X 106 for Lp(a) (11) and 3 X 106 for LDL (33 Calculations. It is well established that the apoproteins of LDL (35) and Lp(a) (14) are not removed from the lipoprotein particle within the plasma.…”

Section: Binding Studiesmentioning

confidence: 99%

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Studies on the role of specific cell surface receptors in the removal of lipoprotein (a) in man.

Krempler¹,

Kostner²,

Roscher³

et al. 1983

J. Clin. Invest.

197

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A B S T R A C T The binding of '25I-lipoprotein (a)[Lp(a)] to cell surface receptors was studied on cultured human fibroblasts. The results were compared with corresponding data obtained with 1251-low density lipoproteins (LDL). Equilibrium binding studies showed that Lp(a) is bound with high affinity by the cell surface receptors. The maximum binding capacity for Lp(a) was 37% lower than for LDL. For Lp(a) and LDL, the Scatchard plots displayed linearity, indicating a single category of binding sites. Half-maximal saturation occurred at a concentration of 9.52±1.04 nM for Lp(a) and 7.76±1.29 nM for LDL. Competition binding experiments revealed that Lp(a) and LDL are nearly equally potent in competing each other for the binding sites. Binding of Lp(a) and LDL were followed by suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Cyclohexanedione treatment of Lp(a) and LDL completely abolished receptor binding. Neither Lp(a) nor LDL were specifically bound by fibroblasts obtained from a patient with homozygous familial hypercholesterolemia (FH).The removal mechanisms for Lp(a) and LDL were further compared by in vivo studies. Radioiodinated Lp(a) and LDL [1431][1432][1433][1434][1435][1436][1437][1438][1439][1440][1441] LDL are nearly identical (6-8). The hexose, hexosamin, and sialic acid content, however, are significantly higher in Lp(a) (9). The main protein constituent of both lipoproteins is apolipoprotein B. Lp(a) has an additional apoprotein that has been termed "specific Lp(a) antigen" (9-11). On agarose gel or cellulose acetate Lp(a) migrates somewhat faster than LDL and has therefore also been described as -pre-fl-lipoprotein" (3,4,12).In spite of these remarkable similarities between Lp(a) and LDL, Lp(a) is not a metabolic product of very low density lipoproteins (VLDL), LDL, or METHODS Binding studiesIsolation of Lp(a) and LDL. The details of the method for isolation of Lp(a) and LDL were described recently (14). Plasma was obtained by plasmapheresis from healthy volunteers who had high levels of Lp(a) as checked by doubleimmunodiffusion using monospecific anti-Lp(a) antibodies (14). The plasma was subjected to sequential ultracentrifugation to obtain density fractions from 1.006 to 1.055 g/ml and from 1.055 to 1.110 g/ml. The densities were adjusted by addition of solid NaCl and checked with a density meter (Anton Paar K. G., Graz). All centrifugal procedures were performed in a Beckman L8-70 centrifuge using a Ti 50.2 rotor (Beckman Instruments, Inc., Fullerton, CA). The supernatants, containing the lipoproteins of the d 1.006-1.055 g/ml and 1.055-1.110 g/ml, were collected by tube slicing. These fractions were concentrated to a volume of -5 ml by dialysis against polyethyleneglycol and then applied to an agarose column (Bio Gel A-Sm, Bio-Rad Laboratories, Richmond, CA). Elution of the lipoproteins was performed with 0.15 M NaCl, pH adjusted to 8.5 by addition of NH40H. During all steps of the isolation procedure, Na2EDTA and NaN3 were present in a concentration of 1 mg/ml...

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Section: Discussionmentioning

confidence: 99%

Section: Binding Studiesmentioning

confidence: 99%

Studies on the role of specific cell surface receptors in the removal of lipoprotein (a) in man.

Krempler¹,

Kostner²,

Roscher³

et al. 1983

J. Clin. Invest.

197

View full text Add to dashboard Cite

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“…[3][4][5][6] has been given by Moore (25).] If the centers of gravity of the contour volume and of pF(r) coincide, the third term on the right-hand side of (3) is zero and a contrast variation series results in the determination of R, and a.…”

Section: Theorymentioning

confidence: 99%

“…Much effort has been devoted to their structural elucidation to provide the basis for an understanding of the various transport and regulatory processes in which LDL is involved (for reviews on LDL structure, see refs. [3][4][5].…”

mentioning

confidence: 99%

Structure of the cholesteryl ester core of human plasma low density lipoproteins: selective deuteration and neutron small-angle scattering.

Laggner¹,

Kostner²,

Degovics³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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The structural arrangement of cholesteryl esters in human plasma low density lipoproteins (LDL) has been studied by selective deuteration and neutron small-angle scattering. LDL were labeled by in vitro exchange with two different kinds of deuterated cholesteryl esters, one labeled in the fatty acyl chain (cholesteryl myristate-d27) and the other in the branched side chain of cholesterol (cholesteryl-25,26,27-d7 oleate). Neutron scattering data from deuterated and protonated LDL were compared to identify the locations of the fatty acyl and cholesterol side chain moieties. Below the thermotropic transition, radii of gyration of 60 A and 70 A were obtained for these two domains, respectively, indicating that the cholesteryl nuclei are situated more distantly from the center than the fatty acyl chains. At 370C, above the thermotropic transition of the cholesteryl esters in LDL, both parts have similar radii of gyration of -56 A. This information is used in a discussion of possible structural models for the apolar lipid core of LDL.Control of cholesteryl ester transport in the bloodstream and control of their catabolism are considered as determining factors in normal or pathological lipid metabolism (1, 2). Normally, most of the circulating cholesteryl esters are contained within lipoprotein particles of the low density class (LDL, Mr = 2-3 x 106). Much effort has been devoted to their structural elucidation to provide the basis for an understanding of the various transport and regulatory processes in which LDL is involved (for reviews on LDL structure, see refs. 3-5).The present knowledge on LDL structure has originated to a large extent from x-ray and neutron small-angle scattering (6-17). In essence, all of these studies concur in indicating a quasi-spherical structure of about 240 A in diameter with an apolar core of neutral lipids, mainly cholesteryl esters, and an amphiphatic shell of protein, phospholipids, and unesterified cholesterol. Deckelbaum et al. (14,15) showed that the apolar lipids within LDL undergo a reversible order-disorder transition in the physiological temperature range. Detailed small-angle scattering analyses (9,16,17) suggested that, below the transition, the core of about 150 A in diameter is formed by concentric shells with a repeat of about 37 A. This order is abolished by heating through the transition range, which in its exact temperature depends on various compositional and structural factors (15,18,19).Although the analogy in thermal behavior to isolated cholesteryl ester mesophases indicated that they are responsible for the ordered structure in the core of LDL, the models proposed so far relied on the structure of pure cholesteryl ester phases-e.g., that of cholesteryl myristate (20, 21). In the search for more direct information, we have undertaken the present neutron small-angle scattering study on LDL isomorphously labeled by cholesteryl esters deuterated either in the fatty acid (I) or in the cholesteroi portion (II) (Fig. 1). The location of these deuterated domains, ...

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“…Although the white appearance of layer 3 for patients R. S. and K. S. contrasted with the yellow color of that of M. S. and normal controls, the chemical composition showed no dramatic difference among them. The composition of M. S., and particularly the higher cholesterol ester content in layer 3, more closely resembled that of an adult's (18). The chemical compositions of layer 5 from patient R. S. and from normal controls were very much alike, suggesting that the patient's HDL was normal.…”

Section: Resultsmentioning

confidence: 74%