scholarly journals Purification of low density lipoprotein receptor from liver and its quantification by anti-receptor monoclonal antibodies

1988 ◽  
Vol 253 (2) ◽  
pp. 409-415 ◽  
Author(s):  
E Gherardi ◽  
N Brugni ◽  
D E Bowyer
Keyword(s):  
Monoclonal Antibody ◽  
Low Density Lipoprotein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Liver Homogenate ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Rabbit Liver ◽  
Receptor Protein ◽  
Low Density ◽  
Liver Protein

The low density lipoprotein (LDL) receptor has been purified to homogeneity from rabbit liver by a combination of DEAE-Sephacel chromatography, LDL-Sepharose 4B chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The receptor protein had a pI of 4.45 and an Mr of 120 x 10(3)-125 x 10(3) in SDS gels under non-reducing conditions. Incubation of the LDL receptor with neuraminidase decreased its Mr to 105 x 10(3)-110 x 10(3) and increased its pI from 4.45 to 5.25. The purified receptor exhibited all the properties of the membrane-bound receptor including Ca2+-dependent binding of rabbit and human LDL but not of methylated LDL or high density lipoprotein. The amount of LDL receptor present in rabbit liver was measured by a quantitative blotting procedure employing a newly developed rat anti-receptor monoclonal antibody. The affinity and specificity of this monoclonal antibody allowed the quantification of the LDL receptor in detergent extracts of liver homogenate, thus eliminating the loss of receptor associated with the preparation of membrane fractions prior to receptor assay. Livers from adult female New Zealand White rabbits contained 149 +/- 13 ng of LDL receptor/mg of liver protein. Administration of pharmacological doses of 17 alpha-ethinyloestradiol raised the concentration of LDL receptor in liver to 312 +/- 25 ng/mg of liver protein.

scholarly journals Detection of the low-density-lipoprotein receptor with biotin-low-density lipoprotein. A rapid new method for ligand blotting

1985 ◽  
Vol 229 (3) ◽  
pp. 785-790 ◽  
Author(s):  
D P Wade ◽  
B L Knight ◽  
A K Soutar
Keyword(s):  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
High Sensitivity ◽  
Receptor Protein ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Cell Extracts ◽  
Density Lipoprotein Receptor ◽  
A New Technique

A new technique has been developed to identify low-density-lipoprotein (LDL) receptors on nitrocellulose membranes, after transfer from SDS/polyacrylamide gels, by ligand blotting with biotin-modified LDL. Modification with biotin hydrazide of periodate-oxidized lipoprotein sugar residues does not affect the ability of the lipoprotein to bind to the LDL receptor. Bound lipoprotein is detected with high sensitivity by a streptavidin-biotin-peroxidase complex, and thus this method eliminates the need for specific antibodies directed against the ligand. The density of the bands obtained is proportional to the amount of pure LDL receptor protein applied to the SDS/polyacrylamide gel, so that it is possible to quantify LDL receptor protein in cell extracts. Biotin can be attached to other lipoproteins, for example very-low-density lipoproteins with beta-mobility, and thus the method will be useful in the identification and isolation of other lipoprotein receptors.

scholarly journals Measurement of the absolute number of functioning low-density lipoprotein receptors in vivo using a monoclonal antibody

1995 ◽  
Vol 305 (3) ◽  
pp. 897-904 ◽  
Author(s):  
C Fitzsimmons ◽  
R Bush ◽  
D Hele ◽  
C Godliman ◽  
E Gherardi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Body Weight ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Absolute Number ◽  
Low Density ◽  
Ldl Receptors ◽  
The Absolute

MAC188 S/S is a monoclonal antibody which can be used in vivo to measure the absolute number of functioning low-density lipoprotein (LDL) receptors in a rabbit. The antibody binds to the extra-cellular domain of the LDL receptor and binding is not blocked by the presence of LDL. When the antibody-receptor complex is internalized, receptor recycling is inhibited for several hours. Thus when saturating doses of MAC188 S/S are administered intravenously, the amount of antibody removed from the blood (minus non-specific removal) is determined solely by the total number of LDL receptors in an animal. In this study MAC188 S/S was used to measure the number of LDL receptors in control rabbits and in animals treated with 17 alpha-ethinyl oestradiol. After treatment (which caused a 47% decrease in plasma cholesterol), receptor-mediated removal of MAC188 S/S from the blood was saturated in both groups following injection of 3.0 mg of antibody per kg body weight. Based on the amount of antibody removed via the LDL receptor at this dose, the total number of accessible LDL receptors was calculated as (2.0 +/- 0.3) x 10(15) receptors per kg body weight in control rabbits and (4.0 +/- 0.4) x 10(15) receptors per kg body weight in oestrogen-treated animals. The number of receptors in various organs was also determined. The monoclonal antibody approach therefore, allows accurate determination of LDL receptor numbers in animals with markedly different concentrations of circulating LDL, conditions in which the use of endogenous ligand would be subject to significant errors.

scholarly journals Immunoprecipitation of the low-density-lipoprotein (LDL) receptor and its precursor from human monocyte-derived macrophages

1986 ◽  
Vol 233 (3) ◽  
pp. 683-690 ◽  
Author(s):  
A K Soutar ◽  
B L Knight
Keyword(s):  
Mononuclear Cells ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Human Monocyte ◽  
Skin Fibroblasts ◽  
Receptor Protein ◽  
Low Density ◽  
Human Mononuclear Cells ◽  
Cell Extracts

Synthesis of the low-density-lipoprotein (LDL) receptor protein by cultured human monocyte-derived macrophages was demonstrated by immunoprecipitation of [35S]methionine-labelled cell extracts with a monoclonal antibody to the bovine adrenal LDL receptor. Although the antibody does not bind to or inhibit binding of 125I-LDL to the LDL receptor on intact fibroblasts, it specifically binds to a protein in extracts of human skin fibroblasts, of Mr approx. 130,000 under non-reducing conditions, that is able to bind LDL. In monocyte-derived macrophages, as in fibroblasts, the receptor is synthesized as a low-Mr precursor that is converted into the mature protein. The half-life of the precursor in human macrophages is approx. 44 min. In cells from two homozygous familial-hypercholesterolaemic subjects, only the precursor form of the receptor is synthesized. Detection of abnormalities of LDL-receptor synthesis in human mononuclear cells may be a useful aid in diagnosis of familial hypercholesterolaemia that is simpler and quicker than methods requiring growth of cultured skin fibroblasts.

scholarly journals Immunoassay of bovine and human low-density-lipoprotein receptors using monoclonal antibodies

1986 ◽  
Vol 238 (2) ◽  
pp. 405-410 ◽  
Author(s):  
B L Knight ◽  
S Preyer ◽  
A K Soutar
Keyword(s):  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Deae Cellulose ◽  
Density Lipoprotein ◽  
Receptor Protein ◽  
Cell Protein ◽  
Human Skin Fibroblast ◽  
Low Density ◽  
Normal Human Skin ◽  
Routine Assay

Two methods are described for the assay of low-density-lipoprotein (LDL) receptor protein based on the binding of receptor to microtitre plate wells coated with a specific monoclonal antibody or with LDL, followed by detection with radioactive antibody that recognizes a different part of the molecule. The two-antibody procedure detected approx. 2 ng of pure bovine receptor at twice background, and there was a linear relationship on a double-logarithm plot between radioactive antibody bound and the amount of receptor added, up to at least 500 ng of receptor protein per well. The procedure using immobilized LDL was less sensitive and the binding of receptor was inhibited by low concentrations of NaCl, which restricted its usefulness for routine assay of tissue extracts. LDL receptor protein could be readily assayed using the two-antibody procedure in normal human skin fibroblast extracts prepared by bulk-elution from small columns of DEAE-cellulose followed by rapid desalting. No radioactive antibody bound with extracts of cells from a receptor-negative familial hypercholesterolaemic subject. The LDL receptor content of normal fibroblasts preincubated with lipoprotein-deficient serum was estimated, using bovine receptor as standard, to be approx. 60 ng of receptor protein/mg of cell protein.

scholarly journals Chemical composition-oriented receptor selectivity of L5, a naturally occurring atherogenic low-density lipoprotein

2011 ◽  
Vol 83 (9) ◽  
pp. 1731-1740 ◽  
Author(s):  
Liang-Yin Ke ◽  
David A. Engler ◽  
Jonathan Lu ◽  
Risë K. Matsunami ◽  
Hua-Chen Chan ◽  
...  
Keyword(s):  
Chemical Composition ◽  
Oxidized Ldl ◽  
Low Density Lipoprotein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Protein Distribution ◽  
Receptor Selectivity

Anion-exchange chromatography resolves human plasma low-density lipoprotein (LDL) into 5 subfractions, with increasing negative surface charge in the direction of L1 to L5. Unlike the harmless L1 to L4, the exclusively atherogenic L5 is rejected by the normal LDL receptor (LDLR) but endocytosed into vascular endothelial cells (ECs) through the lectin-like oxidized LDL receptor-1 (LOX-1). Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional electrophoresis showed that the protein framework of L1 was composed mainly of apolipoprotein (apo) B100, with an isoelectric point (pI) of 6.620. There was a progressively increased association of additional proteins, including apoE (pI 5.5), apoAI (pI 5.4), apoCIII (pI 5.1), and apo(a) (pI 5.5), from L1 to L5. Liquid chromatography data-independent parallel-fragmentation mass spectrometry (LC/MSE) was used to quantify protein distribution in all subfractions. On the basis of weight percentages, L1 contained 99 % apoB-100 and trace amounts of other proteins. In contrast, L5 contained 60 % apoB100 and substantially increased amounts of apo(a), apoE, apoAI, and apoCIII. The compositional characteristics contribute to L5’s electronegativity, rendering it unrecognizable by LDLR. LOX-1, which has a high affinity for negatively charged ligands, is known to mediate the signaling of proinflammatory cytokines. Thus, the chemical composition-oriented receptor selectivity hinders normal metabolism of L5, enhancing its atherogenicity through abnormal receptors, such as LOX-1.

scholarly journals 39-kDa receptor-associated protein (RAP) facilitates secretion and ligand binding of extracellular region of very-low-density-lipoprotein receptor: implications for a distinct pathway from low-density-lipoprotein receptor

1999 ◽  
Vol 341 (2) ◽  
pp. 377-383 ◽  
Author(s):  
Atsushi SATO ◽  
Yoshimi SHIMADA ◽  
Joachim HERZ ◽  
Tokuo YAMAMOTO ◽  
Hisato JINGAMI
Keyword(s):  
Culture Medium ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Receptor Protein ◽  
Low Density ◽  
Vldl Receptor ◽  
Receptor Associated Protein ◽  
Extracellular Region ◽  
Density Lipoprotein Receptor

We have expressed the extracellular regions of the low-density-lipoprotein (LDL) receptor and the very-low-density-lipoprotein (VLDL) receptor, along with the full-length forms of the receptors, in insect cells in a baculovirus system. The extracellular region of the LDL receptor has been secreted successfully into the culture medium, and it retained the capacities of binding 125I-labelled LDL and β-VLDL. In contrast, the extracellular region of the VLDL receptor remained intracellular and it did not bind 125I-β-VLDL. This difference in expression behaviour between the homologous regions of the two receptors suggests that the two receptor systems are different in receptor-protein maturation or protein targeting. Next we developed the co-expression system with 39-kDa receptor-associated protein (RAP). This co-expression facilitated the secretion of the extracellular region of the VLDL receptor into the culture medium and the secreted receptor bound 125I-β-VLDL. The VLDL receptor remaining intracellular that was co-expressed with RAP also showed binding capacity to 125I-β-VLDL, implying that the existence of RAP prevented receptor-protein aggregation or improved protein-folding status of the truncated VLDL receptor. On the other hand, expression of the extracellular region of the LDL receptor was not facilitated by RAP co-expression. Thus RAP plays an essential role in maintenance of the active conformation and secretion of the extracellular region of the VLDL receptor.

scholarly journals Circadian rhythm in hepatic low-density-lipoprotein (LDL)-receptor expression and plasma LDL levels

1994 ◽  
Vol 298 (1) ◽  
pp. 39-43 ◽  
Author(s):  
S Balasubramaniam ◽  
A Szanto ◽  
P D Roach
Keyword(s):  
Low Density Lipoprotein ◽  
Circadian Variation ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Receptor Expression ◽  
Receptor Protein ◽  
Low Density ◽  
Hmg Coa Reductase ◽  
Cultured Fibroblasts ◽  
Coa Reductase

On the basis of studies in vivo and in vitro that involved the use of pharmacological amounts of drugs and hormones or excess cholesterol supplementation, the expression of the low-density lipoprotein (LDL) receptor appears to be tightly coupled to the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity and to extracellular levels of LDL. The present study was undertaken to see how these three entities are regulated under normal physiological conditions over a 24 h period. The results show that, in the rat, hepatic LDL-receptor expression and plasma LDL levels exhibit diurnal periodicity, with a 2-3-fold difference between the peak and trough of each rhythm. Both rhythms showed high inverse correlation (r = -0.86, P < 0.01), plasma LDL levels being lowest at the onset of darkness when LDL-receptor expression was at its peak. The results also showed that the LDL-receptor protein in rat liver has a shorter half-life than that reported for cultured fibroblasts or HepG2 cells. The maximal expression of the LDL receptor occurred several hours before the peak activity of HMG-CoA reductase and appeared not to be influenced by cellular or membrane cholesterol levels during the 24 h cycle. Treatment with dexamethasone increased the LDL-receptor activity significantly at both the lowest and highest points of the rhythm, but the receptor rhythm was still maintained, suggesting that the signal for the circadian variation of the receptor expression is not mediated by adrenal hormones.

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