Native and acetylated low density lipoprotein metabolism in proliferating and quiescent bovine endothelial cells in culture

Lipoprotein binding and metabolism in actively dividing (sparse) and quiescent (confluent) bovine aortic endothelial cells (EC) were compared quantitatively using 125I-labelled lipoproteins. The amounts of receptor-bound low density lipoproteins (LDL) decreased five- to ten-fold as the cultures progressed from sparse to confluent morphology. High affinity receptor-bound LDL levels were extremely low in confluent EC and accounted for the inability of confluent EC to internalize and degrade significant amounts of LDL. Conversely, the amounts of acetylated LDL (acLDL) bound and degraded via distinct sites increased at least five-fold during EC growth to confluence. LDL binding and metabolism in individual cells was assessed by fluorescence microscopy using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labelled lipoproteins or fluorescein-conjugated antibodies. LDL and acLDL bound to the surfaces of sparse EC, at either 4 degrees or 37 degrees C, in a random distribution of fine punctate foci, contrary to a previous report. EC therefore appear to resemble fibroblasts in their distribution of surface LDL receptors. No binding or uptake of LDL was seen in confluent EC. Patterns of acLDL binding and uptake in confluent EC resembled those of LDL in sparse EC. Intracellular LDL and acLDL occurred as perinuclear accumulations of large fluorescent foci in sparse EC. Regeneration experiments were carried out in artificially wounded confluent cultures and renewed LDL receptor activity was shown in actively-dividing cells which had migrated into the "wounded" areas. We conclude that quiescent endothelial cells metabolize little LDL via the LDL-receptor pathway due to a drastically reduced number of receptors in confluent cells. This contrasts with the ability of confluent cells to metabolize relatively large amounts of acLDL via a receptor-mediated mechanism.     

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.     

The insulin-receptor interaction: is the kinetic approach for inferring negative-cooperative site-site interactions valid?

The dissociation of insulin from its receptor is reportedly enhanced when the dissociation is induced by dilution in the presence of insulin. This experiment is frequently conducted when curvilinear Scatchard plots of insulin binding are observed in order to infer negative cooperative site-site interactions amongst insulin receptors. However, when insulin binding to purified liver plasma membranes was measured at 15 degrees C in 50 mM Tris, pH 7.5 containing 0.1% bovine serum albumin and 100 U/ml bacitracin, the insulin binding data was characterised by a linear Scatchard plot and a Hill plot with a slope equal to unity. Thus, under the conditions of this binding assay, insulin apparently bound to a single non-interacting class of homogeneous binding sites. But, despite the apparent absence of cooperative interactions under these specific conditions, the dissociation of receptor-bound insulin was still enhanced when the dissociation of insulin from its receptor was induced by dilution in the presence of insulin. This result cast serious doubt on the validity of inferring negative-cooperative site-site interactions amongst insulin receptors based solely on the observation that the dissociation of receptor-bound insulin is enhanced by dilution in the presence of insulin.     

Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia

Monoclonal antibodies directed against the low density lipoprotein (LDL) receptor have been prepared by immunization of mice with a partially purified receptor from bovine adrenal cortex. Spleen cells from the mice were fused with the Sp2/0-Ag14 line of mouse myeloma cells. The most extensively studied monoclonal antibody, designated immunoglobulin-C7, reacts with the human and bovine LDL receptor, but not with receptors from the mouse, rat, Chinese hamster, rabbit, or dog. 125I-labeled monoclonal antibody binds to human fibroblasts in amounts that are equimolar to 125I-LDL. In fibroblasts from 6 of 8 patients with the receptor-negative form of homozygous familial hypercholesterolemia, which have less than 5% of normal LdL binding, the amount of monoclonal antibody binding was also less than 5% of normal. Fibroblasts from the other two receptor-negative homozygotes bound an amount of monoclonal antibody that was much greater than expected on the basis of LDL binding, suggesting that these two patients produce a structurally altered receptor that binds the antibody, but not LDL. In normal fibroblasts, the receptor-bound monoclonal antibody was taken up and degraded at 37 degrees C at rapid rate similar to that for LDL. Fibroblasts from a patient with the internalization defective form of familial hypercholesterolemia bound the monoclonal antibody, but did not internalize or degrade it. The current data demonstrate the usefulness of monoclonal antibodies as probes for the study of the cellular and genetic factors involved in receptor-mediated endocytosis.     

Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

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.     

Characterization of human adipocyte adenosine receptors

125I-Hydroxyphenylisopropyl adenosine (125I-HPIA) was used to characterize adenosine receptors in human adipocyte plasma membranes. Steady state binding was achieved after 6 h at 37 degrees. Scatchard plots were linear, with a KD of approx. 2.5 nM, and Bmax of 360-1800 fmol/mg protein. (-)N6-phenylisopropyl adenosine (PIA) was a more potent inhibitor of binding than N-ethyl carboxamido adenosine, and (+)PIA was more than 10-fold less potent than (-)PIA, consistent with A1 adenosine receptor binding. Theophylline was a potent inhibitor of binding (IC50 approx. 10 microM). Photoaffinity cross-linking studies demonstrated that the receptor is a single subunit, Mr approx. 43 kDa. The findings demonstrate that the human adipocyte adenosine receptor is similar to the A1 adenosine receptor of rat adipocytes, although its molecular weight is higher, and its affinity for HPIA is lower than that of the rat.     

The expressed human hepatic receptor for low-density lipoproteins differs from the fibroblast low-density lipoprotein receptor

The role of the cellular receptor for the low-density lipoproteins (LDL) in cholesterol transport was initially defined through the study of nonhepatic cells in vitro. Since the liver is central in plasma lipoprotein metabolism, an investigation of hepatic lipoprotein receptors is important for understanding normal lipoprotein transport. Utilizing human hepatic and fibroblast membranes, the characteristics of receptors for LDL from hepatic and nonhepatic tissues were directly compared. Human hepatic membranes reversibly bound LDL within 5 min. Although both fibroblast and hepatic membranes saturably bound LDL at 37 degrees C, the fibroblast LDL receptor affinity (Kd = 2.5 X 10(-8) M) and number (5.5 X 10(12) sites/mg membrane protein) were greater than the hepatic receptor affinity (Kd = 10.8 X 10(-8) M) and number (0.5 X 10(12) sites/mg membrane protein). In contrast to the fibroblast LDL receptor which was unable to bind LDL in the presence of EDTA, the hepatic LDL receptor binding of LDL was only partially blocked by EDTA. The binding of LDL to its hepatic receptor is highly temperature-dependent, and studies utilizing both radiolabeled LDL and colloidal gold-labeled LDL indicate that little, if any, binding of LDL hepatic membranes occur at 0-4 degrees C. The hepatic membrane receptor(s) (Mr approximately equal to 270 000 and 330 000) differ from that of the fibroblast LDL receptor (Mr approximately equal to 130 000) and these proteins are present in hepatic membranes from a patient lacking the fibroblast LDL receptor. These data indicate that an expressed hepatic LDL receptor has unique properties different from those of the fibroblast LDL receptor and that the expressed protein(s) is genetically distinct from the fibroblast receptor.     

Evidence for isomerization during binding of apolipoprotein-B100 to low density lipoprotein receptors.

To determine the kinetics of human low density lipoproteins (LDL) interacting with LDL receptors, 125I-LDL binding to cultured human fibroblasts at 4 degrees C was studied. Apparent association rate constants did not increase linearly as 125I-LDL concentrations were increased. Instead, they began to plateau which suggested that formation of initial receptor-ligand complexes is followed by slower rearrangement or isomerization to complexes with higher affinity. To test this, 125I-LDL were allowed to associate for 2, 15, or 120 min, then dissociation was followed. The dissociation was biphasic with the initial phase being 64-110-fold faster than the terminal phase. After binding for 2 min, a greater percentage of 125I-LDL dissociated rapidly (36%) than after association for 15 min (24%) or 120 min (11%). Neither the rate constants nor the relative amplitudes of the two phases were dependent on the degree of receptor occupancy. Thus, the duration of association, but not the degree of receptor occupancy affected 125I-LDL dissociation. To determine if binding by large LDL, which is predominantly via apolipoprotein (apo) E, also occurs by an isomerization mechanism, the d = 1.006-1.05 g/ml lipoproteins were fractionated by ultracentrifugation. In contrast to small LDL which bound via apoB-100 and whose dissociation was similar to that of unfractionated LDL, large LDL dissociation after 2, 15, or 120 min of binding did not show isomerization to a higher affinity. This suggests that large and small LDL bind by different mechanisms as a result of different modes of interaction of apoE and apoB-100 with LDL receptors.     

Affinity labeling of high-affinity alpha-thrombin binding sites on the surface of hamster fibroblasts

The serine proteinase alpha-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). 125I-labeled alpha-thrombin binds rapidly and specifically to CCL39 cells with high affinity (Kd approximately 4 nM). Binding at 37 degrees C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of alpha-thrombin receptors on CCL39 cells was identified by covalently coupling 125I-alpha-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major alpha-thrombin-binding site of Mr approximately 150 000 revealed as a 125I-alpha-thrombin cross-linked complex of Mr approximately 180 000. Independent of chemical cross-linking, 125I-alpha-thrombin also formed a covalent complex with a minor, 35 000 Mr, membrane component identified as protease nexin. Two derivatives of alpha-thrombin modified at the active site are 1000-fold less than alpha-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native alpha-thrombin, and affinity-labeled the receptor subunit of Mr 150 000. When present in large excess, during incubation of cells with alpha-thrombin, these binding antagonists were ineffective in blocking alpha-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 Mr binding sites that display high affinity for alpha-thrombin do not mediate induction of the cellular mitogenic response.     

Monoclonal antibodies against human low density lipoprotein. Stoichiometric binding studies using Fab fragments

Because of its physical properties, apolipoprotein B (apo B) has remained poorly characterized. In an attempt to elucidate apo B structure, the Fab fragments of 3 different monoclonal anti-human apo B antibodies were tested in a quantitative assay for their binding to human low density lipoprotein (LDL). In each case the assay gave a linear Scatchard plot with a maximum of 1 Fab fragment bound to a single LDL particle. This result favors an LDL model containing a single large Mr apo B protein, which is not composed of multiple, identical, small Mr subunits.     

Absence of positive co-operativity in the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to its cytosolic receptor protein.

The role of positive co-operativity in stabilizing the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the rat hepatic cytosolic TCDD receptor protein (Ah receptor) was investigated. The binding mechanism of TCDD was determined by kinetic means through equilibrium and saturation binding studies, and Scatchard and Hill plot analysis. In all studies, the slope of the Hill plot was close to 1.0, indicating the absence of positive co-operativity. Interpretation of the Scatchard plot was however complicated by the fact that both linear and nonlinear plots were experimentally obtained. The nonlinearity was shown to be an experimental artifact and a consequence not of co-operativity, but of high levels of nonspecific binding. The high level of nonspecific binding could be attributed to: (1) lipophilicity of the TCDD ligand, and (2) inefficient competition of receptor-bound [3H]TCDD. When nonspecific binding was minimized, the Scatchard slope was linear and in agreement with the Hill coefficient, thus indicating the lack of positive co-operativity in the binding of TCDD to the Ah receptor.     

Characterization of the guinea pig adipocyte thyrotropin receptor

125I-TSH binding to porcine thyroid and guinea pig fat resulted in curvilinear Scatchard plots with similar dissociation constants for the high and low affinity binding components. Antibodies from the sera of patients with Graves' disease inhibited binding to the high and low affinity binding components of both tissues. Covalent cross-linking of 125I-TSH to membranes from each tissue resulted in the specific labeling of two protein bands. The guinea pig fat receptor subunits have Mr values of 52,000 and 38,000, whereas the porcine thyroid receptor subunits have values of 46,000 & 35,000. The labeling of the receptor subunits was inhibited by preincubation with Graves' autoantibodies. Despite possessing a different subunit composition, the receptors from these tissues exhibit similar affinity for TSH and share similar antigenic determinants for Graves' autoantibodies.     

A synthetic heparin-mimicking polyanionic compound binds to the LDL receptor-related protein and inhibits vascular smooth muscle cell proliferation

A synthetic heparin-mimicking polyaromatic anionic compound RG-13577 (polymer of 4-hydroxyphenoxy acetic acid and formaldehyde ammonium salt, Mr approximately 5800) exhibits specific binding to vascular smooth muscle cells (SMCs) and inhibits their proliferative response to growth promoting factors. Receptor binding of (14)C-RG-13577 was efficiently competed by apolipoprotein E3 (apoE), lactoferrin, and the LRP (LDL receptor-related protein) receptor associated 39 kDa protein (RAP). Unlike cell surface binding of apoE, binding of RG-13577 to SMCs was not affected by heparin, heparan sulfate degrading enzymes, or low density lipoprotein (LDL). Moreover, wild-type and heparan sulfate-deficient Chinese hamster ovary (CHO) cells, as well as normal- and LDL receptor negative- human skin fibroblasts bind RG-13577, but not apoE, to a similar extent. On the other hand, homozygous mouse embryonic fibroblasts deficient in the LDL receptor-related protein (LRP) expressed a markedly reduced binding of RG-13577 as compared to normal mouse embryonic fibroblasts. These results indicate that RG-13577 and related compounds bind to the LRP receptor on the surface of vascular SMCs. Addition of lactoferrin to cultured SMCs protected the cells against the antiproliferative effect of compound RG-13577, suggesting that this inhibition is mediated by RG-13577 binding to LRP receptors on the SMC surface. Altogether, we have identified a series of synthetic polyaromatic anionic molecules that exhibit specific binding to LRP and thereby exert an antiproliferative effect on vascular SMCs. These compounds are applied to suppress SMC proliferation associated with restenosis and accelerated atherosclerosis.     

Binding of soluble type I collagen molecules to the fibroblast plasma membrane.

Soluble 125I-labeled type I collagen binds to cultured fibroblasts but not to cultured epithelia. The binding of the ligand to fibroblasts is reversible, saturable and highly specific for sequences contained within the helical portions of the alpha1 and alpha2 chains. The amount of ligand bound is dependent upon cell number and ligand concentration. Binding is decreased but measurable at 4 degrees C. The steady state binding is greater at 26 degrees than at 37 degrees C due to a more rapid dissociation of the ligand-acceptor complex at 37 degrees C. The half-life of the complex is 46 min at 37 degrees C and approximately 2.5 hr at 26 degrees C. Scatchard plots of binding data indicate a single class of high affinity binding sites (KD = 1.2 X 10(-11) M) with each fibroblast binding approximately 500,000 molecules at saturation. Pretreatment of fibroblasts with bacterial collagenase, chondroitinase ABC or testicular hyaluronidase does not affect the binding reaction, whereas pretreatment of the cells with phospholipase C increases the amount of ligand bound. Ligand binding is decreased but not abolished after fibroblasts are treated with trypsin concentrations which remove surface fibronectin. Fibroblast monolayers treated with antiserum against fibronectin bind the radiolabeled ligand normally. In contrast to collagen, addition of excess fibronectin does not accelerate the dissociation of bound ligand from fibroblasts. Possible functions for surface-bound collagen are discussed.     

Phorbol esters inhibit the binding of low-density lipoproteins (LDL) to U-937 monocytelike cells

The present study demonstrates that U-937 monocytelike human cells possess specific LDL receptors. 125I-LDL binds at 4 degrees C on the cell surface. The bound molecules are releasable by heparin. The reaction requires Ca2+ and the binding sites are sensitive to proteolysis. Unlabeled LDL compete with 125I-LDL, whereas HDL are ineffective. At 37 degrees C, LDL are internalized and degraded by a chloroquine-sensitive pathway. Tumor-promoting phorbol esters inhibit the binding of 125I-LDL to its receptor on U-937 cells. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, inhibition is 50% at 5 X 10(-9) M of TPA. After removal of phorbol esters, treated cells recover their 125I-LDL-binding activity in 60 min. The inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the number of available LDL receptors rather than a decrease in receptor affinity.     

Characterization of the cell surface receptor for granulocyte-macrophage colony-stimulating factor

125I-labeled recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to characterize receptors specific for this lymphokine on the surface of cells of both myelomonocytic and T-cell origin. GM-CSF binding to these cells was specific and saturable. Equilibrium binding studies revealed that on all cell types examined, GM-CSF bound to a single class of high affinity receptor (1000-5000 receptors/cell) with a Ka of 10(8)-10(9) M-1. More extensive characterization with P388D1 cells showed that binding of GM-CSF was rapid at 37 degrees C with a slow subsequent dissociation rate. Among a panel of lymphokines and growth hormones, only unlabeled natural or recombinant GM-CSF were able to compete for the binding of 125I-GM-CSF to these cells. Affinity cross-linking experiments with the homobifunctional cross-linking reagents disuccinimidyl suberate, disuccinimidyl tartrate, and dithiobis(succinimidyl propionate) resulted in the identification of a receptor protein with a Mr of 130,000 on five out of the seven cell types examined. This protein was extremely sensitive to proteolysis and in the absence of protease inhibitors was degraded to a form with an approximate Mr of 70,000. A receptor protein of Mr 180,000, in addition to the Mr 70,000 protein, was found on bone marrow cells and on P815 cells. The potential tissue-specific molecular heterogeneity associated with the GM-CSF receptor may help to explain some of the diverse biological effects associated with this growth and differentiation factor.     

Specific binding of 125I-labeled human chorionic gonadotropin to gonadal tissue: comparison of limited-point saturation analyses to Scatchard analyses for determining binding capacities and factors affecting estimates of binding capacity

Experiments were conducted to compare gonadotropin binding capacity calculated from limited-point saturation analyses to those obtained from Scatchard analyses, and to test the effects of membrane purity and source of gonadotropin receptors on determining the maximum percentage of radioiodinated hormone bound to receptors (maximum bindability). One- to four-point saturation analyses gave results comparable to results by Scatchard analyses when examining relative binding capacities of receptors. Crude testicular homogenates had lower estimates of maximum bindability of 125I-labeled human chorionic gonadotropin than more purified gonadotropin receptor preparations. Under similar preparation techniques, some gonadotropin receptor sources exhibited low maximum bindability.     

Two classes of receptor specific for sperm-activating peptide III in sand-dollar spermatozoa.

We characterized receptors specific for sperm-activating peptide III (SAP-III: DSDSAQNLIQ) in spermatozoa of the sand dollar, Clypeaster japonicus, using both binding and cross-linking techniques. Analyses of the data obtained from the equilibrium binding of a radiolabeled SAP-III analogueto C. japonicus spermatozoa, using Klotz, Scatchard and Hill plots, showed the presence of two classes of receptors specific for SAP-III in the spermatozoa. One of the receptors (high-affinity) had a Kd of 3.4 nM and 3.4 x 10(4) binding sites/spermatozoon. The other receptor (low-affinity) had a Kd of 48 nM, with 6.1 x 10(4) binding sites/spermatozoon. The Kd of the high-affinity receptor was comparable to the median effective concentration of the intracellular-pH-increasing activity of SAP-III and that of the low-affinity receptor was comparable to the median effective concentration of the cellular-cGMP-elevating activity of the peptide. In addition, Scatchard and Hill plots of the data suggested the existence of positive cooperativity between the high-affinity members. Similar results were also obtained from a binding experiment using a sperm-membrane fraction prepared from C. japonicus spermatozoa. The incubation of intact spermatozoa or sperm plasma membranes with the radioiodinated SAP-III analogue and a chemical cross-linking reagent, disuccinimidyl suberate, resulted in the radiolabeling of three proteins with molecular masses of 126, 87 and 64 kDa, estimated by SDS/PAGE under reducing conditions.     

Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans.

The sulfated glycosaminoglycan, heparin, was found to release 125I-labeled low density lipoprotein (125I-LDL) from its receptor site on the surface of normal human fibroblasts. Measurement of the amount of 125I-LDL released by heparin permitted the resolution of the total cellular uptake of 125I-LDL at 37 degrees C into two components: first, an initial rapid, high affinity binding of the lipoprotein to the surface receptor, from which the 125I-LDL could be released by heparin, and second, a slower process attributable to an endocytosis of the receptor-bound lipoprotein, which rendered it resistant to heparin release. At 4 degrees C the amount of heparin-releasable 125I-LDL was similar to that at 37 degrees C, but interiorization of the lipoprotein did not occur at the lower temperature. The physiologic importance of the cell surface LDL receptor was emphasized by the finding that mutant fibroblasts from a subject with homozygous Familial Hypercholesterolemia, which lack the ability to take up 125I-LDL at 37 degrees C, did not show cell surface binding of 125I-LDL, as measured by heparin release, at either 4 degrees C or 37 degrees C. Although heparin released 125I-LDL from its binding site, it did not release 3H-concanavalin A from its surface receptor, and conversely, alpha-methyl-D-mannopyranoside, which released 3H-concanavalin A, did not release surface-bound 125I-LDL. When added to the culture medium simultaneously with LDL, heparin prevented the binding of LDL to its receptor and hence prevented the LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. The uptake of LDL by fibroblasts is proposed as a model of receptor-mediated adsorptive endocytosis of macromolecules in human cells.     

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

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.