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1.
Metabolism of low-density lipoproteins by cultured hepatocytes from normal and homozygous familial hypercholesterolemic subjects 总被引:1,自引:0,他引:1
J M Hoeg S B Edge S J Demosky T E Starzl T Triche R E Gregg H B Brewer 《Biochimica et biophysica acta》1986,876(3):646-657
The profoundly elevated concentrations of low-density lipoproteins (LDL) present in homozygous familial hypercholesterolemia lead to symptomatic cardiovascular disease and death by early adulthood. Studies conducted in nonhepatic tissues demonstrated defective cellular recognition and metabolism of LDL in these patients. Since mammalian liver removes at least half of the LDL in the circulation, the metabolism of LDL by cultured hepatocytes isolated from familial hypercholesterolemic homozygotes was compared to hepatocytes from normal individuals. Fibroblast studies demonstrated that the familial hypercholesterolemic subjects studied were LDL receptor-negative (less than 1% normal receptor activity) and LDL receptor-defective (18% normal receptor activity). Cholesterol-depleted hepatocytes from normal subjects bound and internalized 125I-labeled LDL (Bmax = 2.2 micrograms LDL/mg cell protein). Preincubation of normal hepatocytes with 200 micrograms/ml LDL reduced binding and internalization by approx. 40%. In contrast, 125I-labeled LDL binding and internalization by receptor-negative familial hypercholesterolemic hepatocytes was unaffected by cholesterol loading and considerably lower than normal. This residual LDL uptake could not be ascribed to fluid phase endocytosis as determined by [14C]sucrose uptake. The residual LDL binding by familial hypercholesterolemia hepatocytes led to a small increase in hepatocyte cholesterol content which was relatively ineffective in reducing hepatocyte 3-hydroxy-3-methylglutaryl-CoA reductase activity. Receptor-defective familial hypercholesterolemia hepatocytes retained some degree of regulatable 125I-labeled LDL uptake, but LDL uptake did not lead to normal hepatocyte cholesterol content or 3-hydroxy-3-methylglutaryl-CoA reductase activity. These combined results indicate that the LDL receptor abnormality present in familial hypercholesterolemia fibroblasts reflects deranged hepatocyte LDL recognition and metabolism. In addition, a low-affinity, nonsaturable uptake process for LDL is present in human liver which does not efficiently modulate hepatocyte cholesterol content or synthesis. 相似文献
2.
Immunoassay of bovine and human low-density-lipoprotein receptors using monoclonal antibodies. 总被引:1,自引:1,他引:0
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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. 相似文献
3.
Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor 总被引:14,自引:0,他引:14
C Koo M E Wernette-Hammond T L Innerarity 《The Journal of biological chemistry》1986,261(24):11194-11201
The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol. 相似文献
4.
Immunoblot analysis of low density lipoprotein receptors in fibroblasts from subjects with familial hypercholesterolemia 总被引:25,自引:0,他引:25
U Beisiegel W J Schneider M S Brown J L Goldstein 《The Journal of biological chemistry》1982,257(21):13150-13156
This paper describes a sensitive method for study of the isoelectric point and molecular weight of immunoreactive low density lipoprotein (LDL) receptors of cultured human fibroblasts. The fibroblast receptors are solubilized with Triton X-100, partially purified by batch elution from DEAE-cellulose, and subjected to two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are transferred electrophoretically to nitrocellulose paper which is then incubated with a mouse monoclonal antibody (IgG-C7) directed against the LDL receptor, followed by an 125I-labeled antibody against mouse IgG. The receptor-bound monoclonal antibody is localized by autoradiography. By this technique, the immunodetectable LDL receptors from normal human fibroblasts migrate as a single spot with an isoelectric point of 4.3 and a Mr of approximately 160,000. In one patient with homozygous familial hypercholesterolemia whose cells fail to bind 125I-labeled IgG-C7, no immunoreactive LDL receptor spot was detected after electrophoresis. We also studied LDL receptors from three homozygotes whose cells bind 125I-IgG-C7, i.e. cross-reacting material-positive mutants. Their immunodetectable receptors were indistinguishable from normal receptors in terms of isoelectric point and molecular weight. Similarly, the receptors from one patient with the internalization-defective form of familial hypercholesterolemia showed normal electrophoretic migration. The immunoblotting technique should prove useful in analyzing structural alterations, if they exist, in LDL receptors from other subjects with cross-reacting material-positive forms of familial hypercholesterolemia. 相似文献
5.
Immunoprecipitation of the low-density-lipoprotein (LDL) receptor and its precursor from human monocyte-derived macrophages. 总被引:2,自引:1,他引:1
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. 相似文献
6.
Low density lipoprotein receptor activity in homozygous familial hypercholesterolemia fibroblasts 总被引:1,自引:0,他引:1
C F Semenkovich R E Ostlund R A Levy S R Osa 《The Journal of biological chemistry》1982,257(21):12857-12865
We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL receptor-negative homozygous familial hypercholesterolemia (FHC). Km and maximum capacity for cell-associated and degraded 125I-LDL were determined by two independent methods, a traditional technique in which increasing amounts of 125I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of 125I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The Km for specific cell-associated 125I-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells have reduced affinity as well as reduced capacity for LDL. The FHC cell receptors share many but not all properties of the normal skin fibroblast LDL receptor. Specific degradation of bound 125I-LDL occurred concomitantly with LDL binding and was greatly reduced by the addition of chloroquine, an inhibitor of lysosomal function. Preincubation of FHC cells with cholesterol or LDL resulted in significant suppression of receptor function. Modification of lysine residues of LDL abolished receptor activity in both normal and FHC cells. Treatment of FHC cells with compactin, a cholesterol synthesis inhibitor, resulted in significant increases in specific 125I-LDL binding and degradation compared to FHC cells without compactin treatment. Normal cells also showed increases in 125I-LDL binding and degradation with compactin treatment, but the mean percentage increase in specific 125I-LDL degradation was significantly greater in FHC cells (strain GM 2000, 160 +/- 18%) than in normal cells (29 +/- 8%). 相似文献
7.
Use of antipeptide antibodies to demonstrate external orientation of the NH2-terminus of the low density lipoprotein receptor in the plasma membrane of fibroblasts
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《The Journal of cell biology》1983,97(5):1635-1640
The low density lipoprotein (LDL) receptor is a member of a class of receptors that bind macromolecules at the cell surface and facilitate their cellular uptake by receptor-mediated endocytosis. The orientation of the LDL receptor in the plasma membrane is unknown. In the current studies the sequence of amino acids at the NH2-terminus of the bovine adrenal LDL receptor was determined, and a synthetic peptide corresponding to amino acids 1-16 was prepared. Antibodies against this peptide were raised in rabbits and were shown by immunoblotting analysis to react specifically with the bovine LDL receptor. The anti- receptor peptide antibodies also bound to the LDL receptor on the outer surface of the plasma membrane of intact human fibroblasts, as visualized by indirect immunofluorescence. Specificity of this binding reaction was confirmed by the observation that the anti-receptor peptide antibodies did not bind to mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack LDL receptors. These data demonstrate that the LDL receptor is oriented in the plasma membrane with its NH2-terminus facing the extracellular surface. 相似文献
8.
Antipeptide antibody against the human low-density-lipoprotein receptor. Characterization and cross-reactivity with bovine lymphocytes.
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G Agnani C Delpierre S Benhamamouch G Torpier C Cachera M Koffigan A Tartar J C Fruchart V Clavey 《The Biochemical journal》1989,263(3):753-760
A dodecapeptide corresponding to the external N-terminal sequence of the human low-density-lipoprotein (LDL) receptor was synthesized. Antibodies raised in rabbits against the peptide were purified and were shown to react specifically with the peptide and with human LDL receptor of fibroblasts, HeLa cells and lymphocytes using binding studies and immunoblotting. By indirect immunogold analysis, antibodies bound to the LDL receptor of human lymphocytes could be revealed as clusters. Anti-receptor peptide immunoglobulins specifically bound to the human HeLa cell's LDL receptor with a lower affinity than LDL (Kd x 3). The anti-receptor peptide immunoglobulins and 125I-labelled-LDL competed with each other for the LDL-receptor sites. Antibodies failed to react with lymphocytes of subjects with the homozygous form of familial hypercholesterolaemia. Cross-reactivity with the dodecapeptide of the bovine LDL receptor was limited, but this cross-reactivity was confirmed by the binding of anti-receptor peptide immunoglobulins to the LDL receptor from bovine lymphocytes. 相似文献
9.
A simple binding assay for the determination of low-density lipoprotein receptors in cell homogenates 总被引:2,自引:0,他引:2
A convenient binding assay has been developed for the determination of low-density lipoprotein (LDL) receptors in homogenates of cultured and freshly-isolated normal and malignant human cells. Cell homogenates were incubated with 125I-labeled LDL and the ligand bound to the homogenate particulates was separated from the unbound ligand by filtration. When the particulates of the homogenates were subsequently incubated with heparin, a fraction of the bound 125I-LDL was released. Previous studies on intact cells have shown that heparin exclusively releases LDL bound to its cell surface receptor. The heparin-sensitive binding of 125I-LDL to cell homogenate particulates represents LDL bound to its cell surface receptor as judged from the following criteria: (a) it was quantitatively similar to the heparin-sensitive binding of 125I-LDL to intact cells, (b) it showed a direct correlation to the receptor-mediated degradation of 125I-LDL by intact cells, (c) no heparin-sensitive binding could be detected in homogenates prepared from normal erythrocytes or from cultured fibroblasts from a patient with homozygous familial hypercholesterolemia (two types of cell lacking LDL receptors), (d) it was dependent on calcium and inhibited by EDTA, (e) it was susceptible to treatment with pronase, and (f) it was heat-labile. The assay developed should be of value in determining the number of LDL receptors in tissues, since it is far less time-consuming and requires less material than currently available methods. 相似文献
10.
Visualization of lipoprotein receptors by ligand blotting 总被引:32,自引:0,他引:32
T O Daniel W J Schneider J L Goldstein M S Brown 《The Journal of biological chemistry》1983,258(7):4606-4611
This paper describes the visualization of the low density lipoprotein (LDL) receptor by ligand blotting. Preparations of detergent-solubilized membranes are subjected to one- or two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, after which the proteins are transferred to nitrocellulose paper. The paper is incubated with native LDL and then with an 125I-labeled antibody against LDL, and the bound antibody is visualized by autoradiography. The success of LDL blotting depends on the omission of sulfhydryl reducing agents from the electrophoresis system. Intrachain disulfide bonds allow the receptor to retain its binding activity even after electrophoresis in the presence of SDS. In identifying LDL receptors, the ligand blotting technique is as sensitive as immunoblotting with a monoclonal antibody against the LDL receptor; it can therefore be used to identify receptors when no anti-receptor antibodies are available. We use this technique to show that the LDL receptor of the rabbit adrenal gland has the same molecular weight as the LDL receptor of the bovine adrenal cortex and human fibroblasts. The ligand blotting technique may be generally applicable for visualization of other plasma membrane receptors after SDS-gel electrophoresis. 相似文献
11.
Tohru Funahashi Yasuko Miyake Akira Yamamoto Yuji Matsuzawa Bun-ichiro Kishino 《Human genetics》1988,79(2):103-108
Summary Mutations of the low density lipoprotein (LDL) receptor in 16 Japanese kindreds with homozygous familial hypercholesterolemia (FH) were studied using an anti-LDL receptor antibody. The LDL receptor mutations in Japanese FH were heterogeneous and included defects in synthesis, posttranslational processing, ligand-binding activity, and internalization of the LDL receptor. Of the 16 kindreds, 10 were receptor-negative and 5, receptor-defective types and 1 was an internalization-defective type with respect to LDL binding. The receptor-negative group was further subdivided into four groups: those with cells producing (i) no immunodetectable receptor (five kindreds); (ii) 160-kd mature receptors, which were quite scarce (two kindreds); (iii) receptors that could not be processed to the mature receptor properly (two kindreds); and (iv) receptors with an apparent molecular weight smaller than normal (one kindred). The last kindred synthesized an about 155-kd mature receptor that was rapidly degraded. This finding is compatible with the low concentration of the cell surface LDL receptors and decreased binding activity for LDL in the cells of this kindred. The receptor-defective group, which could produce a residual amount of functional receptors, exhibited a lower tendency to coronary artery disease than the receptor-negative group. 相似文献
12.
Fibroblasts cultured from the skin of subjects with homozygous familial hyperlipoproteinemia (HFH) internalize and degrade low density lipoproteins at a much lower rate than do fibroblasts from normal subjects. Evidence has been presented that this reflects the absence from such mutant cells of specialized binding sites with high affinity for low density lipoproteins. The specificity of this membrane defect in familial hypercholesterolemia is further supported by the present studies comparing the metabolism of low density lipoproteins (LDL) and high density lipoproteins (HDL) in normal fibroblasts and in fibroblasts from HFH patients. The surface binding (trypsin-releasable (125)I) of (125)I-labeled LDL by HFH cells was approximately 30% of that by normal cells at a concentration of 5 micro g LDL protein per ml. At the same concentration the internalization (cell-associated (125)I after trypsinization) and degradation (trichloroacetic acid-soluble non-iodide (125)I) of (125)I-labeled LDL were less than 10% of the values obtained with normal cells. In contrast, the binding of (125)I-labeled HDL to HFH cells was actually somewhat greater than that to normal cells. Despite this, the internalization and degradation of (125)I-labeled HDL by HFH cells averaged only 70% of that by normal cells. [(3)H]- or [(14)C]Sucrose uptake, a measure of fluid uptake by pinocytosis, was similar in normal and HFH fibroblasts. These findings are consistent with the proposal that fibroblasts from subjects with HFH lack high-affinity receptors for LDL. These receptors do not play a significant role in HDL binding and uptake. Instead, as previously proposed, HDL appears to bind randomly on the cell surface and its internalization is not facilitated by the specific mechanism that internalizes LDL. The small but significant abnormalities in HDL binding and internalization, however, suggest that there may be additional primary or secondary abnormalities of membrane structure and function in HFH cells. Finally, the observed overall rate of uptake of LDL (that internalized plus that degraded) by HFH fibroblasts was considerably greater than that expected from fluid endocytosis alone. This implies that adsorptive endocytosis, associated with binding to low-affinity sites on the cell surface, may play a significant role in LDL degradation by HFH cells, even though it does not regulate endogenous cholesterol synthesis in these cells. 相似文献
13.
S J Mao R E Kazmar J C Silverfield M C Alley K Kluge C G Fathman 《Biochimica et biophysica acta》1982,713(2):365-374
Spleen cells obtained from mice immunized with human plasma low-density lipoproteins (LDL) were fused with mouse myeloma cells. The resulting hybridoma cells secreting immunoglobulin specific for LDL were screened and scored by radioimmunoassay and cloned by multiple limiting dilutions. Immunochemical properties of the monoclonal antibodies were compared with convential mouse serum antibodies. It was found that conventional antibodies precipitated LDL and bound more than 95% of 125I-labeled LDL and the maximal binding was independent of temperature. The monoclonal antibodies were incapable of precipitating LDL and bound a maximum of only 20% of the total 125I-labeled LDL. The maximal binding between monoclonal antibodies and LDL was extremely temperature-dependent. An optimal degree of binding was observed at 4 degrees C, whereas binding at 37 degrees C was only 30% of that achieved at 4 degrees C. Although the binding at 37 degrees C was low, the maximal binding could be re-established following a subsequent incubation at 4 degrees C, suggesting that the antigenic structure of LDL is reversibly modulated at temperatures between 4 and 37 degrees C. Since the orientation of apolipoprotein B in LDL is known to be dynamic at different temperatures, this result suggests that monoclonal antibodies, but not conventional antibodies, are capable of detecting subtle conformational changes in LDL. In addition, we have determined the binding affinity of LDL to monoclonal antibodies and to conventional antibodies. Only monoclonal antibodies showed a linear Scatchard plot, suggesting that the binding was to a single site with a single affinity. The monoclonal antibodies also possessed high specificity and failed to react with porcine LDL, while serum antibodies could recognize both human and porcine LDL. 相似文献
14.
Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor 总被引:14,自引:0,他引:14
L R?nnstrand L Terracio L Claesson-Welsh C H Heldin K Rubin 《The Journal of biological chemistry》1988,263(21):10429-10435
Two monoclonal antibodies against the receptor for platelet-derived growth factor (PDGF) were obtained by immunizing mice with pure PDGF receptor preparations derived from porcine uterus. The antibodies, denoted PDGFR-B1 and PDGFR-B2, both bound to the external domain of the receptor, as demonstrated by indirect immunofluorescence and binding of 125I-labeled antibodies to intact human fibroblasts. Both antibodies precipitated pure 175-kDa 32P-labeled autophosphorylated porcine PDGF receptor as well as a Mr 175,000 glycoprotein from metabolically labeled cells. The monoclonal antibodies did not inhibit binding of 125I-PDGF to human fibroblasts and did not stimulate these cells to undergo mitosis. Both antibodies induced clustering and down-regulation of their antigen. However, this resulted in only a partial loss of cell surface binding sites for PDGF itself, consistent with the conclusion that the monoclonals recognized only one of two or several receptors for PDGF. Clustering and down-regulation were not seen when the cells were incubated with monovalent Fab' fragments of the PDGFR-B2 antibody. The antibodies also stimulated autophosphorylation of pure PDGF receptor, and PDGFR-B2 was shown to stimulate phosphorylation of phosphofructokinase, an exogenous substrate for the PDGF receptor kinase. High concentrations of PDGFR-B2 antibody, or Fab' fragments thereof, failed to enhance the PDGF receptor kinase activity, compatible with the possibility that dimerization was of importance in the antibody-stimulated kinase activity of purified PDGF receptors. 相似文献
15.
M Santillo P Mondola F Santangelo A Belfiore I Tedesco C Laezza R Cerbone A Romano M Bifulco 《Comparative biochemistry and physiology. B, Comparative biochemistry》1992,101(3):489-494
1. BRL-3A cells possess a specific LDL receptor with an apparent mol. wt of 160,000 that binds, with saturation, both human and rat 125I-LDL. 2. Like human fibroblasts, BRL-3A cells also bind, internalize and degrade 125I-hLDL but to a lesser extent. 3. BRL-3A cells also bind the monoclonal antibody against rat liver LDL receptor P1B3. Moreover the LDL receptor activity increases when cells are preincubated with medium containing 5% of LPDS. 4. As with human (h) fibroblasts, treatment of BRL-3A cells with 10(-7) M insulin enhances binding (30%), internalization (18%) and degradation (20%) of 125I-hLDL. 相似文献
16.
Characterization of the low density lipoprotein receptor in membranes prepared from human fibroblasts. 总被引:15,自引:0,他引:15
An ultracentrifugation assay has been developed to measure low density lipoprotein (LDL) receptor activity in membranes prepared from cultured human fibroblasts. The binding site for 125I-labeled LDL in isolated membranes reflected the properties of the LDL receptor previously demonstrated in intact fibroblasts. It exhibited high affinity (Kd approximately 4 microgram of LDL protein/ml), specificity (LDL approximately 400-fold more effective than high density lipoprotein in competing with 125I-LDL for the binding site), dependence on calcium, and susceptibility to destruction by pronase. The number of LDL receptors detected in the in vitro membrane binding assay was similar to the number detected in intact cells. The number of receptors was reduced in membranes from fibroblasts that were grown in the presence of 25-hydroxycholesterol plus cholesterol and in fibroblast membranes from a subject with homozygous familial hypercholesterolemia, two situations in which the number of LDL receptors in intact fibroblasts is known to be reduced. The availability of a membrane binding assay that faithfully reflects the properties of the physiologic LDL receptor of intact cells should permit the characterization of this receptor in organs from intact humans and animals. 相似文献
17.
Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts 总被引:16,自引:7,他引:9
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《The Journal of cell biology》1981,90(3):595-604
The visible wavelength excited fluorophore 3,3'- dioctadecylindocarbocyanine iodide (Dil[3]) was incorporated into human low density lipoprotein (LDL) to form the highly fluorescent LDL derivative dil(3)-LDL. Dil(3)-LDL binds to normal human fibroblasts and to human fibroblasts defective in LDL receptor internalization but does not bind to LDL receptor-negative human fibroblasts at 4 degrees C or 37 degrees C. It is internalized rapidly at 37 degrees C by normal fibroblasts and depresses the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in a manner similar to that of LDL. It is prevented from binding to the LDL receptor by an excess of unlabeled LDL or by heparin sulfate. Identical distributions of dil(3)- LDL are observed on cells by either indirect immunofluorescence with fluorescein-labeled antibody or directly by dil(3) fluorescence. Upwards of 45 molecules of dil(3) are incorporated per molecule of LDL without affecting binding to the receptor. This labeling renders individual molecules visible by their fluorescence and enables the derivative to be used in dynamic studies of LDL-receptor motion on living fibroblasts by standard fluorescence techniques at low LDL receptor density. Observations with this derivative indicate that the LDL-receptor complex is immobilized on the surface of human fibroblasts but, when free of this linkage, undergoes a Brownian motion consistent with theory. 相似文献
18.
To assess the relationship of apoB structures in different species of animals, the expressions of apoB epitopes in the sera or plasmas of 23 different mammalian species and one marsupial, and on the low density lipoprotein (LDL) from three species of apes, six species of monkeys, and eight non-primates were measured in competitive radioimmunoassays. The abilities of the sera or LDL to compete with 125I-labeled human LDL for binding to seven monoclonal antihuman LDL antibodies immobilized on microtiter plates were determined. LDL of apes bound to most antibodies, while monkey LDL bound to two or three antibodies. Other mammalian LDL bound only weakly to two of the antibodies or to none. The two monoclonal antibodies binding the LDL of more species were those antibodies which also inhibited the binding to and degradation of LDL by human fibroblasts. The rank order of binding of the LDL of a given species to the antibodies correlated with the rank order inhibition of binding and degradation of 125I-labeled human LDL in the human fibroblast system. This suggests that epitopes spatially located near the recognition site of apoB for cellular receptors have a greater tendency to be conserved. 相似文献
19.
Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate 总被引:1,自引:0,他引:1
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《The Journal of cell biology》1987,105(6):2551-2558
Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I- thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation. 相似文献
20.
Stoichiometric binding of low density lipoprotein (LDL) and monoclonal antibodies to LDL receptors in a solid phase assay 总被引:3,自引:0,他引:3
The current paper describes a solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane-spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells. 相似文献