Effects of sodium butyrate on the membrane glycoconjugates of murine sarcoma virus-transformed rat cells.

The temporal relationship between butyrate-induced cellular flattening of murine sarcoma virus-transformed rat cells (MSV-NRK) and alterations in certain surface-associated biochemical markers of transformation, e.g., surface glycopeptides, glycolipids, fibronectin, hexose uptake, and cell-substrate adhesion was examined. The induction of elevated levels of the ganglioside GM3 and of a GDla-like ganglioside were observed to precede or to parallel cellular flattening. Likewise, enhanced incorporation of radioisotopically labeled fucose into a novel fucose-containing component, i.e., glucopyranosyl (1 leads to 3) fucopyranosyl-threonine, was also observed to occur at an early stage of cellular flattening. In contrast, a shift in the molecular weight distribution of trypsin-sensitive, surface fucopeptides was observed to occur at a late stage of cellular flattening. Moreover, surface fibronectin was not detectable in the butyrate-flattened MSV-NRK cells despite the fact that the cells manifested significantly enhanced cell-substrate adhesion. Thus, butyrate appears to be a useful tool for understanding the sequential changes associated with expression of the transformed phenotype of MSV-NRK cells.

Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein.

Acetylated-low density lipoprotein (Ac-LDL) is taken up by macrophages and endothelial cells via the "scavenger cell pathway" of LDL metabolism. In this report, aortic and microvascular endothelial cells internalized and degraded 7-15 times more [125I]-Ac-LDL than did smooth muscle cells or pericytes. Bound [125I]-Ac-LDL was displaced by unlabeled Ac-LDL, but not unmodified LDL. The ability to identify endothelial cells based on their increased metabolism of Ac-LDL was examined using Ac-LDL labeled with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil-Ac-LDL). When cells were incubated with 10 micrograms/ml Dil-Ac-LDL for 4 h at 37 degrees C and subsequently examined by fluorescence microscopy, capillary and aortic endothelial cells were brilliantly fluorescent whereas the fluorescent intensity of retinal pericytes and smooth muscle cells was only slightly above background levels. Dil-Ac-LDL at the concentration used for labeling cells had no effect on endothelial cell growth rate. When primary cultures of bovine adrenal capillary cells were labeled with 10 micrograms/ml of Dil-Ac-LDL for 4 h at 37 degrees C, then trypsinized and subjected to fluorescence-activated cell sorting, pure cultures of capillary endothelial cells could be obtained. Utilizing this method, large numbers of early passage microvascular endothelial cells can be obtained in significantly less time than with conventional methods.

Hepatic overexpression of bovine scavenger receptor type I in transgenic mice prevents diet-induced hyperbetalipoproteinemia.

Hepatic scavenger receptors (SR) may play a protective role by clearing modified lipoproteins before they target the artery wall. To gain insight into this hypothesized function, transgenic mice expressing hepatic bovine SR (TgSR) were created and studied when fed chow, and during diet-induced hyperlipidemia. SR overexpression resulted in extensive hepatic parenchymal cell uptake of fluorescently labeled acetylated human low density lipoprotein (DiI ac-hLDL) and a twofold increase in 125I-acetylated-LDL clearance. Food intake and cholesterol absorption was indistinguishable between control and TgSR mice. In chow-fed mice, lipoprotein cholesterol was similar in control and TgSR mice. However, on a 3-wk high fat/cholesterol (HFHC) diet, the rise in apoB containing lipoproteins was suppressed in TgSR+/- and TgSR+/+ mice. The rise in HDL was similar in control and TgSR+/- mice, but significantly elevated in the TgSR+/+ mice. Overall, on chow, the ratio of apo-B containing lipoprotein cholesterol to HDL cholesterol was similar for all groups (control = 0.33; TgSR+/- = 0.32; TgSR+/+ = 0.38). However, after 3 wk on the HFHC diet, this ratio was markedly higher in control (2.34 +/- 0.21) than in either TgSR+/- (1.00 +/- 0.24) or TgSR+/+ (1.00 +/- 0.19) mice. In TgSR+/- mice, hepatic cholesteryl esters were reduced by 59%, 7 alpha-hydroxylase mRNA levels were elevated twofold, and a significant increase in fecal bile acid flux was observed after the 3-wk HFHC diet. These results suggest SR may play a protective role in liver by preventing diet-induced increases in apoB containing lipoproteins.

Distinct murine macrophage receptor pathway for human triglyceride-rich lipoproteins.

Murine P388D1 macrophages have a receptor pathway that binds human hypertriglyceridemic very low density lipoproteins (HTG-VLDL) that is fundamentally distinct from the LDL receptor pathway. Trypsin-treated HTG-VLDL (tryp-VLDL), devoid of apolipoprotein (apo)-E, fail to bind to the LDL receptor, yet tryp-VLDL and HTG-VLDL cross-compete for binding to P388D1 macrophage receptors, indicating that these lipoproteins bind to the same sites. The specific, high affinity binding of tryp-VLDL and HTG-VLDL to macrophages at 4 degrees C is equivalent and at 37 degrees C both produce rapid, massive, curvilinear (receptor-mediated) triglyceride accumulation in macrophages. Ligand blots show that P388D1 macrophages express a membrane protein of approximately 190 kD (MBP190) that binds both tryp-VLDL and HTG-VLDL; this binding is competed by HTG-VLDL, trypsinized HTG-VLDL, and trypsinized normal VLDL but not by normal VLDL or LDL. The macrophage LDL receptor (approximately 130 kD) and cellular uptake of beta-VLDL, but not MBP 190 nor uptake of tryp-VLDL, are induced when cells are exposed to lipoprotein-deficient medium and decreased when cells are cholesterol loaded. Unlike the macrophage LDL receptor, MBP 190 partitions into the aqueous phase after phase separation of Triton X-114 extracts. An anti-LDL receptor polyclonal antibody blocks binding of HTG-VLDL to the LDL receptor and blocks receptor-mediated uptake of beta-VLDL by P388D1 cells but fails to inhibit specific cellular uptake of tryp-VLDL or to block binding of tryp-VLDL to MBP 190. Human monocytes, but not human fibroblasts, also express a binding protein for HTG-VLDL and tryp-VLDL similar to MBP 190. We conclude that macrophages possess receptors for abnormal human triglyceride-rich lipoproteins that are distinct from LDL receptors in ligand specificity, regulation, immunological characteristics, and cellular distribution. MBP 190 shares these properties and is a likely receptor candidate for the high affinity uptake of TG-rich lipoproteins by macrophages.

A simple and rapid anti-ligand enzyme immunoassay for visualization of low-density lipoprotein membrane receptors.

Triton X-114 was used to solubilize the membrane proteins of bovine adrenal cortex and human leukocytes. The solubilized membrane proteins were subjected to electrophoresis and transblotted to nitrocellulose paper and incubated with LDL/acetyl-LDL. The combination of peroxidase-conjugated second antibody and 4-chloronaphthol/H2O2 allowed rapid development of colored bands where LDL or acetyl-LDL bound to electroblotted proteins. The ELISA is highly sensitive and efficient for screening a large number of samples and avoids the need for a continuous supply of radiolabeled antibodies and autoradiography.

Metabolism of normal and modified low-density lipoproteins by macrophage cell lines of murine and human origin.

Four murine macrophage-like continuous cell lines (P388D1, J774.1, RAW 264.7, and PU5-1.8) and two human cell lines displaying macrophage-monocyte characteristics (HL-60, U-937) have been examined for their ability to degrade both normal and acetylated low-density lipoproteins. All of these cell lines, except PU5-1.8, were demonstrated to have LDL receptors that were induced 2-5-fold by preincubation in lipoprotein-deficient serum. Metabolism of dextran sulfate-LDL complexes by all lines except PU5-1.8 was observed. Three cell lines, P388D1, J774.1 and RAW 264.7, while exhibiting individual differences in their metabolism of acetyl-LDL, all processed acetyl-LDL in a fashion qualitatively analogous to that by murine peritoneal macrophages and human monocytes. Cell lines PU5-1.8, U-937 and HL-60 did not bind or degrade significant quantities of acetyl-LDL. In P388D1 cells, metabolism of acetyl-LDL exhibited time and concentration dependence, was reversibly inhibited by chloroquine, blocked by fucoidan and dextran sulfate, and was calcium independent. Approximately 4 X 10(5) receptors, with an apparent Kd of 3 X 10(-8) M, were present on P388D1 cells. P388D1 cells metabolized 30% as much acetyl-LDL as murine peritoneal macrophages at 37 degrees C and bound 60% as much at 4 degrees C. Chemical measurement demonstrated a 250-fold increase in the cholesteryl ester content of P388D1 cells over 96 h. The accumulation of cholesteryl esters was reversible in the presence of HDL3 and involved continuous hydrolysis and reesterification. These lines represent a convenient resource for examining the metabolism of chemically modified lipoproteins, for isolation of cell mutants, and for isolation of specific lipoprotein receptors.

Fluorescence assay of the specificity of human plasma and bovine liver phospholipid transfer proteins.

The specificities of a human plasma and bovine liver phospholipid transfer protein were studied using a fluorescence assay based on the transfer of pyrenyl phospholipids. This method was used previously to determine the mechanism of spontaneous transfer of phospholipids between model lipoproteins (Massey, J.B., Gotto, A.M., Jr. and Pownall, H.J. (1982) Biochemistry 21, 3630-3636). The pyrenyl phospholipids varied in the headgroup moiety; pyrenyl phosphatidylcholines contained different fatty acyl chains in the sn-1 position. Model high-density lipoproteins (R-HDL) consisting of apolipoprotein A-I and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were used as donor and acceptor particles. As previously shown, the bovine liver protein mediated the transfer of only phosphatidylcholine. In contrast, the human plasma protein transferred all species studied which included a phosphatidylserine, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, galactosylcerebroside, and a diacylglycerol. The activity of these transfer proteins was only slightly affected by changes in the acyl chain composition of the transferring lipid. Pyrenyl and radioactive ([3H]POPC) phospholipids were transferred with equal rates by the human transfer protein, suggesting that this protein has similar binding characteristics for pyrenyl and natural phospholipids. Spontaneous phospholipid transfer occurs by the aqueous diffusion of monomeric lipid where the rate is highly dependent on fatty acyl chain composition. In this study, no correlation between the rate of spontaneous transfer and protein-mediated transfer was found. The apparent Km values for R-HDL and low-density lipoprotein (LDL), when used as acceptors, were similar when based on the number of acceptor particles. The apparent Vmax for the bovine liver protein was identical for R-HDL and LDL but for the plasma protein Vmax was slightly higher for R-HDL. These results suggest that, like the bovine liver protein, the plasma protein functions as a phospholipid-binding carrier that exchanges phospholipids between membrane surfaces. The assay of lipid transfer proteins by pyrenyl-labeled lipids is faster and easier to perform than other current methods, which require separation of donor and acceptor particles, and is suitable for studies on the function and mechanism of action of lipid transfer proteins.

Measurement and prediction of the rates of spontaneous transfer of phospholipids between plasma lipoproteins.

The purpose of this report is to develop a correlation between the hydrophobicity of a phospholipid as measured by reversed-phase high-performance liquid chromatography and its rate of spontaneous transfer and to use this correlation to predict the rate of transfer of any homologous lipid from any lipoprotein. We have studied the mechanism of transfer of a series of fluorescent or radiolabeled phospholipids among natural and reassembled serum lipoproteins. Fluorescent phosphatidylcholines included those with 9-(1-pyrenyl)nonanoic acid in the sn-2 position and lauric, myristic, palmitic, stearic, oleic or linoleic acid at sn-1. The radioactive phosphatidylcholines contained [3H]oleic acid in the sn-2 position and lauric, myristic, or palmitic acid at sn-1. The kinetics of transfer of the pyrene-labeled lipid were followed by changes in the excimer fluorescence, and that of the radioactive lipids by separation of the donor (lipid-apolipoprotein recombinant) from the acceptor (single bilayer vesicles) on a column of Sephacryl S-200. The retention time of each lipid was measured by high-performance hydrophobic chromatography through a Waters radially compressed C18 column eluted with 75% isopropanol and 25% triethylammonium phosphate (0.15 M). A linear relationship was observed between the rate-constant of transfer and the retention time which suggest that the rate of desorption of phosphatidylcholines from lipoproteins and vesicles is controlled predominately by the hydrophobic effect. For a homologous series of lipids, the rate of transfer can be predicted from retention times obtained from hydrophobic chromatography. The kinetics of transfer of 1-lauroyl-2-[9-(1-pyrenyl)nonanoyl] phosphatidylcholine between isolated human serum lipoproteins exhibits a linear correlation between the transfer half-time and the size of the donor lipoproteins. As a consequence, transfer from very-low-density lipoprotein is 10-times slower than that observed from high-density lipoproteins. The observed correlations between phospholipid transfer rates and both the Stokes radius of the donor and the retention time of the phospholipid on a hydrophobic column permit one to calculate the rate of transfer of homologous molecules between lipid-protein complexes. The results predict that the spontaneous transfer of phospholipids between plasma lipoproteins would be too slow to be a physiologically important phenomena.

Spontaneous and plasma factor-mediated transfer of pyrenyl cerebrosides between model and native lipoproteins.

A series of pyrenyl glucocerebrosides was synthesized by reacylation of psychosine with pyrene-labeled fatty acids having 3-11 methylene units. When incorporated into model high-density lipoproteins consisting of dimyristoylphosphatidylcholine-apolipoprotein A-II complexes and incubated with unlabeled complexes, these lipids exhibited spontaneous transfer. Half times of transfer varied from 1.5 min to 365 min at 37 degrees C. The logarithm of the rate of transfer was linearly related to the number of fatty acyl methylene units and HPLC retention time. Transfer occurred by passage of lipid monomers through the aqueous phase. Spontaneous transfer of the glycolipids also occurred when they were incorporated into native high-density lipoproteins. Rates of transfer between native high-density lipoprotein particles were higher than those observed between model high-density lipoprotein particles. A partially purified lipid exchange protein from plasma, as well as unfractionated lipoprotein-deficient serum, stimulated the transfer of fluorescent glycolipid between model high-density lipoprotein or native high-density lipoprotein and low-density lipoprotein 2-24 fold. The protein also stimulated the transfer of tritiated ganglioside GM3 between native low-density lipoprotein and high-density lipoprotein. This protein may play a role in glycolipid exchange in vivo.

Oxidized low-density lipoproteins inhibit endothelial cell proliferation by suppressing basic fibroblast growth factor expression.

BACKGROUND: Hyperlipidemia inhibits proliferation of endothelial cells (ECs) in culture and angiogenesis in vivo and in arterial explants. Elucidation of the mechanisms may suggest novel therapies against atherosclerosis. METHODS AND RESULTS: Basic fibroblast growth factor (bFGF) expression and mitogenic effects were assessed in bovine aortic ECs incubated with oxidized LDL (ox-LDL). Compared with native LDL and lipoprotein-free controls, ox-LDL reduced bFGF mRNA levels in a time- and concentration-dependent manner, 100 microg/mL producing a maximum reduction of 40% to 50% within 24 to 48 hours. There were commensurate reductions in intracellular and extracellular bFGF concentrations, DNA and total RNA syntheses, and cell replication. FGF receptor 1 and beta-actin mRNA levels were unchanged. Ox-LDL accelerated bFGF mRNA degradation in actinomycin D-treated cells. However, inhibition of bFGF expression by ox-LDL was attenuated by cyclohexamide, indicating a requirement for continuous new protein synthesis for posttranscriptional destabilization. Reduced syntheses of DNA and total RNA were completely restored by bFGF but not by vascular endothelial growth factor. Inhibition of total RNA synthesis achieved by exposing cells to a bFGF-neutralizing antibody was similar in magnitude to that induced by ox-LDL. CONCLUSIONS: Cytotoxic effects of ox-LDL on ECs are attributable in part to suppression of bFGF expression.

Differential regulation of cyclooxygenase-2 (COX-2) mRNA stability by interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in human in vitro differentiated macrophages.

Cyclooxygenase-2 (COX-2) is a highly inducible gene in macrophages by pro-inflammatory cytokines. A major mechanism for cytokine-induced COX-2 expression is stabilization of COX-2 mRNA. In this study, we examined the induction of COX-2 expression by interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in human primary in vitro differentiated macrophages. IL-1 beta (5 ng/mL) or TNF-alpha (1 ng/mL) induced up to an approximately 40-fold increase of COX-2 mRNA in macrophages during a 2 to 2.5-hr incubation. Run-off experiments demonstrated that cytokine stimulation had only a mild effect on the COX-2 transcription rate (approximately 10-40% increase). The translation blocker cycloheximide (CHM) (10 mg/mL) superinduced COX-2 mRNA during 2 hr of incubation and further stabilized the COX-2 mRNA (T1/2 > 4 hr). The CHM-superinduced COX-2 mRNA was subject to a rapid degradation after removal of CHM (T1/2 < 1 hr). Both IL-1 beta and TNF-alpha stabilized cytokine-induced COX-2 mRNA (T1/2 > or = 2 hr). Maximal stabilization of COX-2 mRNA after a short-term stimulation required the continued presence of IL-1 beta in the medium. Long-term treatment of TNF-alpha destabilized the induced COX-2 mRNA. Cells simultaneously treated with both IL-1 beta and TNF-alpha had a reduced induction of COX-2, IL-1 beta, and IL-6 mRNA. In transcription-arrested cells, the translation blocker puromycin affected the TNF-alpha-induced stabilization and destabilization of COX-2 mRNA, but not the IL-1 beta-induced stabilization. The studies suggest that positive and negative regulation of mRNA stability may play a major role in cytokine-mediated COX-2 induction in human macrophages. TNF-alpha may play both pro-inflammatory and protective roles during inflammation by regulation of pro-inflammatory gene transcripts.

Interaction of LDL and Lp[a] with human skin fibroblasts.

We have studied the interaction of LDL and Lp[a] with fibroblasts. Our studies suggest that Lp[a] does not effectively compete with LDL for binding to the LDL receptor, and does not efficiently suppress the activity of the intracellular enzyme HMG-CoA reductase. However, Lp[a-], formed by reduction of the disulfide bond between apo[a] and apoB, behaves much like homologous LDL, whether or not apo[a] is removed from the mixture, and in spite of the fact that one or more apoB disulfides may also have been cleaved. In our studies we also noted that Lp[a] often enhanced binding of 125I-LDL by fibroblasts. Further investigation has suggested that this interaction is time-dependent. Experiments in receptor-negative fibroblasts indicate that the enhancement is not related to the presence of the LDL receptor; however, it is inhibited by the removal of calcium from the medium. The presence of sialic acid at millimolar concentrations in the medium inhibits much of the Lp[a]-enhanced binding of 125I-LDL to the cells. These studies suggest that Lp[] may in some way enhance LDL binding to cells, perhaps via interaction with cell surface glycosaminoglycans or proteoglycans or with collagen.

Solubilization and purification of an acetyl-LDL binding membrane protein from human leukocytes.

An acetyl-LDL receptor in the human leukocyte membrane might have some importance in the generation of foam cells in the atherosclerotic lesion. We show purification of a human leukocyte membrane protein which exhibits ac-LDL-specific binding. Our attempts to solubilize this protein from the leukocyte's membrane fraction involved the usage of the non-ionic detergents Triton X-114 and beta-D-Octylglucoside. Our scheme for the purification of the acetyl-LDL binding protein included ion exchange chromatography and affinity chromatography. Following solubilization the ac-LDL binding activity was assayed by ligand blotting, demonstrating polyvinylsulfate sensitive binding of ac-LDL to a single, acidic 280 000 D membrane protein in the crude membrane fraction.

Binding characteristics of reduced hepatic receptors for acetylated low-density lipoprotein and maleylated bovine serum albumin.

The binding characteristics of reduced hepatic membrane proteins for acetylated low-density lipoprotein (acetyl-LDL) and maleylated bovine serum albumin (Mal-BSA) have been examined. Two receptor activities were extracted from hepatic membranes in the presence of octyl beta-D-glucoside and beta-mercaptoethanol, and were separated by chromatography on Mal-BSA-Sepharose 4B. The receptors were revealed by ligand blotting. The active binding proteins had apparent molecular masses of 35 and 15 kDa in SDS/polyacrylamide gels. Equilibrium studies with protein-phosphatidylcholine complexes indicated that the reduced 35 kDa protein expresses two binding sites for Mal-BSA and one for acetyl-LDL, whereas the 15 kDa protein-phosphatidylcholine complex binds 131I-Mal-BSA and 131I-acetyl-LDL with a 4:1 stoichiometry. 131I-Mal-BSA binding was linear with both proteins, with a Kd of 4.8 nM at the 35 kDa protein and a Kd of 5.6 nM at the 15 kDa protein. The 35 kDa protein displayed saturable binding of 131I-acetyl-LDL with a Kd of 5 nM; the 15 kDa binding protein bound 131I-acetyl-LDL with a Kd of 2.3 nM. A 85 kDa protein was obtained by Mal-BSA-Sepharose chromatography when the hepatic membranes had been solubilized with Triton X-100 in presence of GSH/GSSG. This protein displayed saturable 131I-Mal-BSA binding with a Kd of 30 nM and 131I-acetyl-LDL binding with a Kd of 6.5 nM. The 131I-Mal-BSA binding capacity was four times higher than that of 131I-acetyl-LDL. Competition studies with the 35 kDa, 15 kDa and 85 kDa proteins binding Mal-BSA, acetyl-LDL, formylated albumin and polyanionic competitors provide evidence for the existence of more than one class of binding sites at the reduced binding proteins.

In situ labelling of vascular endothelium with fluorescent acetylated low density lipoprotein.

Acetylated low density lipoprotein is metabolized by a receptor-mediated process in endothelial cells. We have used the lipoprotein labelled with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate to localize endothelial cells lining blood vessels. Following intravenous injection of the labelled lipoprotein, the vascular sinusoids and all other hepatic blood vessels were clearly labelled in cryostat sections of mouse liver. The endothelium of other organs such as brain, kidney, and testis was also brightly labelled. In addition, the lipoprotein was used to label the endothelium of bovine aorta, the vasculature in the chick chorioallantoic membrane, and the vessels in a growing murine melanoma. These results demonstrate that the fluorescent-labelled lipoprotein can be used for in situ labelling of the endothelium from large as well as small blood vessels in a variety of species.

Murine macrophage tumors are a source of a 260,000-dalton acetyl-low density lipoprotein receptor.

Subcutaneous injection of murine macrophage cell line P388D1 into syngeneic DBA/2 produced tumors, which upon solubilization with 40 mM octyl glucoside contained acetylated low density lipoprotein binding activity. The tumor-derived receptor specifically bound acetylated low density lipoprotein with an affinity of approximately 3 X 10(-8) M but did not bind low density lipoprotein or high density lipoprotein. It was identical in binding specificity, affinity, and Pronase sensitivity to the receptor in intact cells or that obtained from solubilized cultured cell membranes. Partial purification of the receptor was achieved by solubilizing tumors with 1% Triton X-100 followed by chromatography on polyethyleneimine cellulose. After elution with a NaCl gradient in the presence of octyl glucoside and association with liposomes, a 287-fold purification of the receptor was achieved. The receptor was identified by specific ligand blotting as a 260,000-dalton protein having a pI of approximately 6.0. Binding to the receptor by acetylated low density lipoprotein, malondialdehyde-modified low density lipoprotein, and maleic anhydride-modified serum albumin was demonstrated by ligand blotting. A single receptor protein can, therefore, account for the binding of multiple types of charge-modified lipoprotein and nonlipoprotein ligands to the macrophage cell surface.

Binding of acetylated low density lipoprotein and maleylated bovine serum albumin to the rat liver: one or two receptors?

The liver is the major organ involved in clearance of acetylated low density lipoprotein (acetyl-LDL) and maleylated serum albumin (Mal-BSA). Quantitative analysis of the hepatic uptake by sequential scintigraphy in rats shows that the hepatic uptake capacity for Mal-BSA is at least 15 times larger than for acetyl-LDL particles. A membrane-associated M approximately 250,000 daltons hepatic receptor for acetyl-LDL and Mal-BSA was 1450-fold purified from total membrane by Triton X-114 solubilization, chromatography on polyethylenimine cellulose and gel filtration. This receptor incorporated into liposomes displayed a saturable binding of [131I]Mal-BSA with a dissociation constant Kd = 15 nM and to [131I]acetyl-LDL with a dissociation constant Kd = 0.9 nM. The binding of both ligands was sensitive to poly(vinyl sulfate). The purified scavenger receptor system has a binding capacity for [131I]Mal-BSA 20 times larger than for [131I]acetyl-LDL. This is similar to the maximal removal capacity of the rat liver for both ligands in vivo. Binding studies with Mal-BSA, acetyl-LDL and anti-idiotypic receptor antibodies as competitors for [131I]Mal-BSA and [131I]acetyl-LDL binding demonstrate that [131I]Mal-BSA and [131I]acetyl-LDL compete for a common binding site. However, not all of the Mal-BSA binding sites are capable of interacting with acetyl-LDL.

Evidence in liver for a disulphide-linked scavenger receptor containing a binding site for acetylated low-density lipoprotein and maleylated bovine serum albumin.

Membranes from rat liver were analysed under reducing conditions. The components of the soluble membranes responsible for the binding of acetylated low density lipoprotein (acetyl-LDL) and maleylated bovine serum albumin (Mal-BSA) were chromatographed on a polyethyleneimine-cellulose column and subsequently separated by gel electrophoresis. For both ligands a major binding protein (Mr = 35,000) was revealed by ligand blotting. A minor protein (Mr greater than 67,000) exhibited little binding. The Scatchard plot of the 131I-Mal-BSA binding data of the 35 kDa protein was linear, with a Kd of 17.3 nM. High concentrations of acetyl-LDL competed for half of the 131I-Mal-BSA binding. Excessive Mal-BSA competed for all the visible acetyl-LDL binding. The findings indicate the existence, in the reduced hepatic membrane, of a 35 kDa protein that has two binding sites for 131I-Mal-BSA and one binding site for acetyl-LDL.