Comparison of R- and S-form lipopolysaccharides fractionated from Escherichia coli UKT-B lipopolysaccharide in pyrogen and Limulus tests

Different LPS was shown to have a relatively different proportion of O-specific chain-less (R-form) LPS by polyacrylamide gel electrophoresis (PAGE) with sodium deoxycholate (DOC). By using DOC-PAGE, S-form LPS having O-chain with approximately 11 repeating units on average (S-Fr) and O-chain-less LPS (R-Fr) were separated from Escherichia coli UKT-B S-form LPS. Significantly stronger pyrogenicity was observed in R-Fr than in S-Fr when measured on the weight basis. Similar result was observed in Limulus test. Comparing biological activities of different S-form LPS, attention should be given to the amounts of co-existing R-form LPS.     

Specific intracellular hyaluronic acid binding to isolated rat hepatocytes is membrane-associated

Intact isolated rat hepatocytes show a small amount of specific 125I-labeled hyaluronic acid (HA) binding. However, in the presence of digitonin, a very large increase in the specific binding of 125I-HA is observed. Chondroitin sulfate, heparin and dextran sulfate were as effective as unlabeled HA in competing for 125I-HA binding to permeabilized hepatocytes, indicating that the binding sites may have a general specificity for glycosaminoglycans. After rat hepatocytes had been homogenized in a hypotonic buffer, more than 98% of the 125I-HA binding activity could be pelleted by centrifugation at 100,000 x g for 1 h. Mild alkaline treatment of hepatocyte membranes did not release 125I-HA binding activity, suggesting that the HA binding site is an integral membrane molecule. Furthermore, trypsin treatment of deoxycholate-extracted membranes destroyed the binding activity, as assessed by a dot-blot assay. This suggests that a protein component in the membrane is necessary for 125I-HA binding activity. Rat fibrinogen could be a possible candidate for the HA binding activity because HA binds specifically to human fibrinogen (LeBoeuf et al. (1986) J. Biol. Chem. 261, 12 586). Also, fibrinogen can be found in a quasi-crystalline form in rat hepatocytes and could be pelleted with the membranes. Rat fibrinogen was not responsible for the 125I-HA binding activity, since (1) purified rat fibrinogen did not bind to 125I-HA, and (2) immunoprecipitation of rat fibrinogen from hepatocyte extracts did not decrease the 125I-HA binding of these extracts. We conclude that the internal HA binding sites are membrane- or cytoskeleton-associated proteins and are neither cytosolic proteins nor fibrinogen.     

Selection of peptides that bind to the core oligosaccharide of R-form LPS from a phage-displayed heptapeptide library

To characterize common sites within the core oligosaccharide of the R-form lipopolysaccharide (LPS), we screened peptides from a phage-displayed heptapeptide library by using the most truncated form of R-LPS, Re-LPS (S. Typhimurium SL1165) as a ligand. After three rounds of biopanning/amplification and subsequent screening by phagemid enzyme-linked immunosorbent assay (ELISA), we selected three distinct clones that bind to the ligand LPS. We characterized the binding sites of the three clones by ELISA and thin-layer chromatography immunostaining and found that the three clones bind the two Re-LPSs (SL1165 and S. Minnesota Re595) and Rb2-LPS. In addition, one of the clones also bound to S-form LPS (S. Enteritidis). Current data show that those clones bind to common carbohydrate structure(s) expressed in the core oligosaccharides of those LPS samples.     

Binding of bacterial lipopolysaccharide to histocompatibility-2-complex proteins of mouse lymphocytes.

The membrane binding sites for lipopolysaccharide (LPS) were isolated by affinity chromatography of the solubilized membranes prepared from 125I-labeled mouse B-cells and T-cells on an affinity adsorbent prepared by coupling Salmonella minnesota R595 LPS to activated Sepharose 4B. The membrane proteins bound to the affinity adsorbent and eluted with 1.0% Triton X-100 were analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecylsulphate. These membrane proteins were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, were identified in the eluate from the B-cell membranes. The histocompatibility-2-complex proteins (H-2D, H-2K and Ia antigens) were also found to be binding sites for LPS on both B-cells and T-cells.     

Staining of O-specific polysaccharide chains of lipopolysaccharides with ruthenium red

S-form lipopolysaccharides (LPS) from Klebsiella strain LEN-1 (O3: K1-) and from Salmonella minnesota strain 1114 were positively stained with ruthenium red, whereas R-form LPS from Klebsiella strain LEN-111 (O3-: K1-) and Ra, Rb1, RcP+, Rd1P-, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by such treatment. From these results it was concluded that ruthenium red stains the O-specific polysaccharide chains of LPS. The appearance of stained preparations of S-form LPS suggested that the material responsible for this positive staining corresponded to the surface projections which were seen by the negative staining technique as attached to the ribbon-like structures and spherules of the LPS.     

Binding properties of high-density lipoprotein subfractions and low-density lipoproteins to rabbit hepatocytes

Primary cultures of rabbit hepatocytes which were preincubated for 20 h in a medium containing lipoprotein-deficient serum subsequently bound, internalized and degraded 125I-labeled high-density lipoproteins2 (HDL2). The rate of degradation of HDL2 was constant in incubations from 3 to 25 h. As the concentration of HDL2 in the incubation medium was increased, binding reached saturation. At 37 degrees C, half-maximal binding (Km) was achieved at a concentration of 7.3 micrograms of HDL2 protein/ml (4.06 X 10(-8)M) and the maximum amount bound was 476 ng of HDL2 protein/mg of cell protein. At 4 degrees C, HDL2 had a Km of 18.6 micrograms protein/ml (1.03 X 10(-7)M). Unlabeled low-density lipoproteins (LDL) inhibited only at low concentrations of 125I-labeled HDL2. Quantification of 125I-labeled HDL2 binding to a specific receptor (based on incubation of cells at 4 degrees C with and without a 50-fold excess of unlabeled HDL) yielded a dissociation constant of 1.45 X 10(-7)M. Excess HDL2 inhibited the binding of both 125I-labeled HDL2 and 125I-labeled HDL3, but excess HDL3 did not affect the binding of 125I-labeled HDL3. Preincubation of hepatocytes in the presence of HDL resulted in only a 40% reduction in specific HDL2 receptors, whereas preincubation with LDL largely suppressed LDL receptors. HDL2 and LDL from control and hypercholesterolemic rabbits inhibited the degradation of 125I-labeled HDL2, but HDL3 did not. Treatment of HDL2 and LDL with cyclohexanedione eliminated their capacity to inhibit 125I-labeled HDL2 degradation, suggesting that apolipoprotein E plays a critical role in triggering the degradative process. The effect of incubation with HDL on subsequent 125I-labeled LDL binding was time-dependent: a 20 h preincubation with HDL reduced the amount of 125I-labeled LDL binding by 40%; there was a similar effect on LDL bound in 6 h but not on LDL bound in 3 h. The binding of 125I-labeled LDL to isolated liver cellular membranes demonstrated saturation kinetics at 4 degrees C and was inhibited by EDTA or excess LDL. The binding of 125I-labeled HDL2 was much lower than that of 125I-labeled LDL and was less inhibited by unlabeled lipoproteins. The binding of 125I-labeled HDL3 was not inhibited by any unlabeled lipoproteins. EDTA did not affect the binding of either HDL2 or HDL3 to isolated liver membranes. Hepatocytes incubated with [2-14C]acetate in the absence of lipoproteins incorporated more label into cellular cholesterol, nonsaponifiable lipids and total cellular lipid than hepatocytes incubated with [2-14C]acetate in the presence of any lipoprotein fraction. However, the level of 14C-labeled lipids released into the medium was higher in the presence of medium lipoproteins, indicating that the effect of those lipoproteins was on the rate of release of cellular lipids rather than on the rate of synthesis.     

Recognition of heptoses and the inner core of bacterial lipopolysaccharides by surfactant protein d

Lipopolysaccharides (LPS) of Gram-negative bacteria are important mediators of bacterial virulence that can elicit potent endotoxic effects. Surfactant protein D (SP-D) shows specific interactions with LPS, both in vitro and in vivo. These interactions involve binding of the carbohydrate recognition domain (CRD) to LPS oligosaccharides (OS); however, little is known about the mechanisms of LPS recognition. Recombinant neck+CRDs (NCRDs) provide an opportunity to directly correlate binding interactions with a crystallographic analysis of the binding mechanism. In these studies, we examined the interactions of wild-type and mutant trimeric NCRDs with rough LPS (R-LPS). Although rat NCRDs bound more efficiently than human NCRDs to Escherichia coli J-5 LPS, both proteins exhibited efficient binding to solid-phase Rd2-LPS and to Rd2-LPS aggregates presented in the solution phase. Involvement of residues flanking calcium at the sugar binding site was demonstrated by reciprocal exchange of lysine and arginine at position 343 of rat and human CRDs. The lectin activity of hNCRDs was inhibited by specific heptoses, including l-glycero-alpha-d-manno-heptose (l,d-heptose), but not by 3-deoxy-alpha-d-manno-oct-2-ulosonic acid (Kdo). Crystallographic analysis of the hNCRD demonstrated a novel binding orientation for l,d-heptose, involving the hydroxyl groups of the side chain. Similar binding was observed for a synthetic alpha1-->3-linked heptose disaccharide corresponding to heptoses I and II of the inner core region in many LPS. 7-O-Carbamoyl-l,d-heptose and d-glycero-alpha-d-manno-heptose were bound via ring hydroxyl groups. Interactions with the side chain of inner core heptoses provide a potential mechanism for the recognition of diverse types of LPS by SP-D.     

Determination of the structural domain of ApoAI recognized by high density lipoprotein receptors.

There is good evidence that high density lipoprotein (HDL) interacts with high affinity sites present on hepatocytes. The precise nature of the ligand recognized by putative HDL receptors remains controversial, although there is a consensus that apolipoprotein AI (apoAI) is involved. This suggestion would be strengthened if a biologically active site demonstrating a high affinity for the receptor could be isolated. Cyanogen bromide fragments (CF) of apoAI (CF1-CF4) were complexed with phospholipid, and their ability to associate with the receptor was compared in various binding studies. Careful analysis of the concentration-dependent association of 125I-labeled dimyristoyl phosphatidylcholine (DMPC) recombinants to rat liver plasma membranes revealed high and low affinity binding components. As all DMPC recombinants displayed the low affinity binding component, it was postulated that this interaction was independent of the protein present in the particle and may well represent a lipid-lipid or lipid-protein association with the membranes. Only 125I-labeled CF4.DMPC displayed a high affinity binding component with similar Kd and Bmax (8 x 10(-9) M, 1.6 x 10(-12) mol/mg plasma membrane protein) to that of 125I-labeled AI.DMPC (7 x 10(-9), 1.4 x 10(-12) mol/mg plasma membrane protein). Similarly, egg yolk phosphatidylcholine complexes containing CF4 (CF4.egg PC) showed higher affinity binding than CF1-egg yolk phosphatidylcholine complexes confirming the results obtained with DMPC complexes. Furthermore, ligand blotting studies showed that only 125I-labeled CF4.DMPC associated specifically with HB1 and HB2, two HDL binding proteins recently identified in rat liver plasma membranes. We conclude that a region within the carboxyl-terminus of apoAI is responsible for the interaction with putative HDL receptors present in rat liver plasma membranes.     

A saturable, high-affinity binding site for human low density lipoprotein on freshly isolated rat hepatocytes

Freshly isolated rat hepatocytes bind the solely apolipoprotein B-containing human low density lipoprotein (LDL) with a high-affinity component. After 1 h of incubation less than 30% of the cell-associated human LDL is internalized and no evidence for any subsequent high-affinity degradation was obtained. Scatchard analysis of the binding data for human 125I-labeled LDL indicates that the high-affinity receptor for human LDL on rat hepatocytes possesses a Kd of 2.6 x 10(-8)M, while the binding is dependent on the extracellular Ca2+ concentration. Competition experiments indicate that both the apolipoprotein B-containing lipoproteins (human LDL and rat LDL) as well as the apolipoprotein E-containing lipoproteins (human HDL and rat HDL) do compete for the same surface receptor. It is concluded that hepatocytes freshly isolated from untreated rats do contain, in addition to the earlier described rat lipoprotein receptor which does not interact with human apolipoprotein B-containing LDL, a high-affinity receptor which interacts both with solely apolipoprotein B-containing human LDL and apolipoprotein E-containing lipoproteins.     

Purification and fractionation of lipopolysaccharide from gram-negative bacteria by hydrophobic interaction chromatography

By hydrophobic interaction chromatography on octyl-Sepharose, lipopolysaccharide (LPS) of Escherichia coli Re mutant and of wild-type smooth-form (S-form) Salmonella typhimurium and Salmonella abortus equi is fractionated according to increasing amount of fatty acids. Thereby a fractionation of S-form LPS according to the length of the O-polysaccharide chain also occurs, because with increasing of fatty acids there is a decrease in the mean length of the O-polysaccharide chain from approximately 30 to 4 repeating units. Molecular species of Re-mutant LPS contain four 3-hydroxytetradecanoyl residues in addition to which dodecanoic, tetradecanoic and possibly hexadecanoic acid, appear in this sequence. Among the molecular species of S-form LPS, dodecanoic, tetradecanoic and hexadecanoic acids appear in the same order, but in contrast to Re-mutant LPS a significant fraction of S-form LPS contains less than four 3-hydroxytetradecanoyl residues. Hydrophobic interaction chromatography also proved an effective one-step purification procedure of LPS as was shown with a crude preparation from S-form S. typhimurium.     

Binding of alpha 2-macroglobulin to hepatocytes: mechanism of in vivo clearance

The binding of 125I-labeled human alpha 2-macroglobulin-methylamine to adult rat hepatocytes in primary culture was studied at 4 degrees C. Cells which had been in culture for 4 hours exhibited steady state ligand binding after 1 hour, a receptor number of 22,400 receptors per cell, and a dissociation constant of 0.6 nM. Adult rat hepatocytes exhibited a significant decrease in receptor number with increased time in primary culture with less than 10% of the initial number of receptors remaining after 2 days (p less than 0.01). In autopsy studies of mice injected intravenously with 125I-labeled alpha 2-macroglobulin-methylamine, greater than 90% of the cleared ligand was found in the liver. Autoradiography of the liver demonstrated that 80% of the ligand was cleared by hepatocytes. It is concluded that the hepatocytes are the primary pathway for clearance from the circulation of receptor recognized alpha 2-macroglobulin.     

Synthesis and biochemical characterization of a photoactivatable, iodinatable, cleavable bacterial lipopolysaccharide derivative

A method for the synthesis of a photoactivatable, iodinatable, and thiol-cleavable derivative of bacterial lipopolysaccharide (LPS) is described using sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3'-dithiopropionate. The method described is applicable to LPS from both smooth and rough bacteria. Evidence is presented that the coupling reaction occurs primarily to phosphoethanolamine residues localized to the inner core region of the LPS. Radioiodination of the derivatized LPS results in a product with a specific activity of 1.8-2.5 microCi/micrograms. Experiments comparing the activity of native and derivatized S-form LPS suggest that the synthesis has not introduced major alterations in the biological properties of the LPS. The feasibility of this derivatized LPS as a molecular probe to investigate LPS binding targets in biological systems is suggested by experiments showing ultraviolet light-dependent cross-linking, thiol-dependent cleavage, and subsequent transfer of radioiodine to both monoclonal anti-LPS antibody and bovine serum albumin. The latter interaction has been demonstrated to be highly selective in protein mixtures containing serum albumin in solution with LPS.     

Consequences of 125I-labeled insulin degradation by hepatocytes on the interpretation of receptor binding studies

The association of ¹²⁵I-labeled insulin with hepatocytes was assayed by filtration or microcentrifugation. Assay by centrifugation resulted in a greater amount of retained radioactive label throughout the course of association of ¹²⁵I-labeled insulin with hepatocytes. Similarly, saturation experiments assayed by microcentrifugation suggested greater binding than filtration. During dissociation, cells isolated by centrifugation released a greater amount of rapid-dissociating radioactive label. Control experiments of [³H]-inulin exclusion with cell pellets, which were isolated during microcentrifugation, demonstrated that the difference between the methods was not due to extracellular trapping of radioactivity. Therefore, the data suggested that there was more low-affinity retention when binding was assayed by centrifugation than filtration. The integrity of the ¹²⁵I-labeled insulin extracted from hepatocytes was determined by column chromatography. A substantially greater proportion of the extracted radioactivity was fragments of ¹²⁵I-labeled insulin in cells isolated by centrifugation. It is suggested that the extensive washing of the cells during filtration removes more fragments than does centrifugation. During dissociation, the low-affinity component of radioactivity, which was observed in the centrifugal assay, resulted from the transient retention of insulin fragments. The extensive degradation of insulin, which was assayed by either method, and the differences observed between these methods, should be considered in the interpretation of binding experiments with cells.     

Analysis of the 2-keto-3-deoxyoctonate (KDO) region of lipopolysaccharides isolated from non-01 Vibrio cholerae 05R

Phosphorylated 2-keto-3-deoxyoctonate (KDO) has been detected in the strong-acid hydrolysates of lipopolysaccharides (LPS) of family Vibrionaceae including Vibrio cholerae. Structural analysis of LPS isolated from a rough mutant of non-01 V. cholerae 05 by dephosphorylation, periodate oxidation and methylation analysis revealed that the inner core region of the LPS molecule contains only one mole of KDO in contrast to enteric Gram-negative bacterial LPS, and that the phosphate group on the KDO molecule resides in the C4 position, while the site of binding of KDO to heptose, a constituent of the distal part of the inner core region, is the C5 position as in the enteric bacterial LPS.     

Metabolism of low-density lipoproteins by cultured hepatocytes from normal and homozygous familial hypercholesterolemic subjects

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.     

16-kilodalton heparin binding (fibroblast) growth factor type one appears in a stable 40-kilodalton complex after receptor-dependent internalization

Incubation of 16-kDa 125I-labeled heparin binding (acidic fibroblast) growth factor type one (HBGF-1) with human hepatoma cells and normal rat hepatocytes resulted in the appearance of a stable 125I-labeled complex with an apparent molecular mass of 40 kDa. The complex could be isolated with specific antibodies against HBGF-1. In contrast to membrane receptor-bound 125I-HBGF-1, the complex was resistant to dissociation by detergents, acid, heat, and reducing or denaturing agents. Formation of a stable complex did not require treatment with cross-linking agents. Appearance of the 40-kDa complex was dependent on time, temperature, and enriched culture medium. Conditions that enhanced or reduced display of specific HBGF-1 membrane receptor sites enhanced or reduced the appearance of the 40-kDa complex. Dansylcadaverine, chloroquine, and staurosporine blocked the appearance of the 40-kDa complex concurrent with the blockage of internalization of the receptor-bound HBGF-1. Two-dimensional gel electrophoretic analysis, metabolic labeling with L-[35S]cysteine, and recovery of 16-kDa HBGF-1 from the 40-kDa complex after base treatment suggest involvement of a 24-kDa cellular protein in the complex formation. These results suggest a potentially novel receptor-dependent pathway for metabolism of HBGF-1.     

Identification of dipeptidyl peptidase IV as a protein shared by the plasma membrane of hepatocytes and liver biomatrix

The histotypic organization of liver parenchyma involves specific intercellular contacts and interaction of hepatocytes with supporting biomatrix. Evidence from this laboratory identified a peptide (Hep105, apparent Mr 105 000) that is shared by the plasma membrane of rat hepatocytes and rat liver biomatrix. This report identifies Hep105 as a peptide component of dipeptidyl peptidase IV (DPPIV; EC 3.4.14.-). A monoclonal antibody (MAb 236.3) was shown to immunoprecipitate DPPIV from non-ionic detergent extracts of surface-labeled 125I hepatocytes. The immunoprecipitate contained two 125I-labeled peptides: Hep105 and a peptide of apparent Mr 150000 (Hep150). Proteolysis of 125I-labeled Hep105 and Hep150 by Staphylococcus aureus V8 protease yielded essentially identical patterns of 125I-labeled peptide degradation products, indicating that Hep105 and Hep150 are structurally related. Only Hep150 exhibited DPPIV activity on transblot analysis, an observation that is consistent with the interpretation that it is the monomeric form of the enzyme. Heating (100 degrees C, 5 min) of purified Hep150 in the presence of sodium dodecylsulfate (SDS) resulted in its conversion to Hep105 and the disappearance of any demonstrable enzymatic activity. 3H-labeled diisopropyl fluorophosphate was incorporated into Hep105, indicating that Hep105 contains the active site for DPPIV. Purified rat liver biomatrix was shown to possess significant DPPIV activity. Taken together, these data indicate that Hep105 s a peptide component of DPPIV.     

Characteristics of the binding of high-density lipoprotein3 by intact cells and membrane preparations of rat intestinal mucosa

We have investigated the binding of high-density lipoprotein (HDL3, d = 1.12-1.21 g/ml), and apolipoprotein E-deficient human and rat HDL, obtained by heparin-Sepharose affinity chromatography, to intact cells and membrane preparations of rat intestinal mucosal cells. Binding of 125I-labeled HDL3 to the basolateral plasma membranes was characterised by a saturable, specific process (Kd = 21 micrograms of HDL3 protein/ml, Bmax = 660 ng HDL3 protein/mg membrane protein) and E-deficient human HDL demonstrated a similar affinity for the binding site. The basolateral plasma membranes isolated from proximal and distal portion of rat small intestine showed similar binding affinities for HDL3, whereas the interaction of HDL with brush-border membranes was characterised by mainly nonspecific and nonsaturable binding. The binding of 125I-labeled HDL3 to basolateral plasma membranes was competitively inhibited by unlabeled HDL3 but less efficiently by unlabeled human LDL. The putative HDL receptor was not dependent on the presence of divalent cations but was markedly influenced by temperature and sensitive to pronase treatment. We have also demonstrated, using whole intestinal mucosal cells, that lysine and arginine-modified HDL3 inhibited binding of normal 125I-labeled HDL3 to the same extent as normal excess HDL3. These data suggest that basolateral plasma membranes of rat intestinal mucosal cells possess a specific receptor for HDL3 which contains mainly apolipoprotein A-I and A-II, and the mechanisms of recognition of HDL3 differ from those involved in binding to the B/E receptor.     

Ceruloplasmin receptors in liver cell suspensions are limited to the endothelium

The interaction of ceruloplasmin (CP) with isolated liver cell suspensions was studied using 125I-labeled and latex minibead-derivatized CP. Fractionation of liver cell suspensions was done using metrizamide gradient centrifugation. In crude liver cell suspensions only endothelial cells, but not hepatocytes and Kupffer cells bound the minibead probe. The binding was specific and inhibited by excess native CP. These results were confirmed using 125I-CP combined with cell fractionation technique. Kinetic data, obtained from the latter system, indicated a dissociation constant (Kd) of 1 X 10(-7) M and the number of receptors to be 5.7 X 10(5) per endothelial cell. The exclusive binding of CP to liver endothelium suggests that this cell may mediate the hepatocytes uptake of CP and is, therefore, a crucial element of the tissue-blood barrier.