Specific High Affinity Binding Sites Research Articles

Atrial natriuretic factor receptors in cultured renomedullary interstitial cells

To gain further insight on the cell types that may mediate the effects of atrial natriuretic factor (ANF) in the renal medulla, we determined the distribution and function of biological (B) and clearance (C) receptors of ANF in renomedullary interstitial cells (RMIC). Studies were performed in the 3rd-17th passages of RMIC obtained from a primary culture of the rat renal medulla. Electron microscopy of the cultured cells showed the typical morphological features of RMIC "in vivo," including prominent lipid droplets. RMIC have a very high density of high-affinity specific binding sites of ANF-(1-28) [23,000 sites/cell; dissociation constant (Kd) = 50 pM]. There was only minimal binding of C-ANF-(4-23) (less than 2,500 sites/cell), a specific ligand of C-ANF receptors. ANF-(1-28) markedly increased guanosine 3',5'-cyclic monophosphate (cGMP) from 1.3 +/- 0.3 to 106 +/- 22 pmol cGMP/10(6) cells [50% effective dosage (ED50) = 1.2 nM]. The effect of ANF-(1-28) on cGMP was nearly additive to that of sodium nitroprusside and was not potentiated or antagonized by C-ANF-(4-23). The density of guanylate cyclase-coupled B-ANF receptors and the ANF-induced increase in cGMP in RMIC are higher than those reported to date in other target cells. This suggests that RMIC may mediate some of the known effects of ANF in the renal medulla.

Insulin-secreting β-cells possess specific receptors for interleukin-1β

The effect of the cytokine interleukin-1β on the insulin secretory responsiveness of single β-cells (HIT-T15) was investigated. In the short-term, IL-1β induced a dosage-dependent stimulation of insulin release. In contrast, in the long-term, IL-1β, inhibited both basal and secretagogue-stimulated insulin secretion. We also demonstrate the simultaneous presence of specific high and low affinity binding sites for IL-1β on β-cells. IL-1β, which has been implicated in the pathogenesis of insulin-dependent diabetes, may therefore mediate its opposing effects on β-cells through a specific plasma membrane receptor.

Binding of 3H-spiperone to human peripheral lymphocytes: Absence of stereospecific high-affinity binding

The binding of 3H-spiperone to human peripheral lymphocytes (PBL) was characterized. The (+)-butaclamol displaceable binding of 3H-spiperone was not saturable, and both (+) and (-)-butaclamol were equally potent in displacing 3H-spiperone binding. We did not find evidence for the existence of a high-affinity specific binding site of 3H-spiperone on human PBL.

Characterization of Endothelin Receptors and Effects of Endothelin on Diacylglycerol and Protein Kinase C in Retinal Capillary Pericytes

Retinal capillary pericyte is a cell type selectively lost in early diabetic retinopathy. The physiological function of pericytes is not yet clearly identified, although it probably has contractile properties. We determined the specific binding of endothelin 1, a 21-amino acid peptide with potent vasoconstrictive action, and the stimulation of diacylglycerol/protein kinase C (DAG/PKC) pathway in cultured retinal capillary pericytes by endothelin. A single specific binding site for 125I-labeled endothelin was identified, with an apparent Kd of 1.3 nM and a maximal binding capacity of approximately 1-2 x 10(5) sites/cell. Endothelin (100 nM) increased total cellular DAG content by 15% at 5 min and 24% at 10 min. When pericytes were labeled isotopically with [3H]glycerol, endothelin stimulated [3H]DAG formation by 100% at 10 min and 88% at 30 min. After 10 min of endothelin treatment, PKC activities were increased by 60 and 100% in the membranous and cytosolic pools, respectively. We conclude that bovine retinal capillary pericytes possess numerous high-affinity specific binding sites for endothelin that mediate the action of endothelin by the stimulation of the DAG/PKC pathway in pericytes. These findings suggest that endothelin is a regulator of the contractile properties of pericytes, which may be adversely affected in diabetic retinopathy.

Androgen receptors, androgen‐dependent proliferation, and 5α‐reductase activity of small‐cell lung cancer cell lines

We investigated the influence of testosterone on binding to established small-cell lung cancer (SCLC) cell lines and were able to show specific high-affinity and low-capacity binding sites in some cell lines with a typical receptor size, using sucrose density gradient centrifugation. In addition, we demonstrated marked growth stimulation with testosterone and dehydrotestosterone using different androgen-receptor-positive small-cell lung cancer cell lines. This growth stimulation could be counteracted by the addition of anti-androgens like cyproterone acetate or flutamide. The presence of 5 alpha-reductase activity, 17 beta-hydroxysteroid-dehydrogenase and 3 alpha-hydroxysteroid-dehydrogenase activities could be demonstrated, suggesting testosterone metabolism of small-cell lung cancer cell lines.

Effect of Non-Solubilizing SDS Concentrations on High Affinity Ca2+ Binding and Steady State Phosphorylation by Inorganic Phosphate of the Sarcoplasmic Reticulum ATPase

In this investigation low, non-solubilizing concentrations of the strong anionic detergent SDS were used to perturbate the interaction of Ca2+ and Pi with their respective binding domains on the sarcoplasmic reticulum Ca-transport ATPase. Rising SDS concentrations produce a two-step decline of Ca2+-dependent ATP hydrolysis. At pH 6.15, SDS differently affects high affinity Ca2+ binding and phosphorylation by inorganic phosphate and releases the "mutual exclusion" of these two ligand binding steps. The degree of uncoupling is considerably more pronounced in the presence of 20% Me2SO. The reduction of Ca2+ binding by SDS is demonstrated to be a result of decreased affinity of one of the two specific high affinity binding sites and of perturbation of their cooperative interaction. Higher SDS partially restores the original high Ca2+ affinity but not the cooperativity of binding. Phosphorylation exhibits a higher SDS sensitivity than Ca2+ binding: Increasing SDS competitively inhibits and then completely abolishes phosphoenzyme formation. Thus, SDS binds to the phosphorylation domain, evidently involving the Lys352 residue of the ATPase molecule; this is accompanied by a more unspecific concentration-dependent SDS effect, probably mediated by hydrophobic force, which, finally, suppresses phosphorylation. Me2SO does neither qualitatively affect the SDS-dependent chemical properties of the vesicular material nor the SDS-dependent perturbation of the investigated reaction steps.

Transmembrane transport of fatty acids in the heart

Although fatty acid uptake by the myocardium is rapid and efficient, the mechanism of their transmembrane transport has been unclear. Fatty acids are presented to the plasma membrane of cardiomyocytes as albumin complexes within the plasma. Since albumin is not taken up by the cells, it was postulated that specific high affinity binding sites at the sarcolemma may mediate the dissociation of fatty acids from the albumin molecules, before they are transported into the cells. In studies with a representative long-chain fatty acid, oleate, it was in fact shown that fatty acids bind with high affinity to isolated plasma membranes of rat heart myocytes revealing a KD of 42 nM. Moreover, a specific membrane fatty acid-binding protein (MFABP) was isolated from these membranes. It had a molecular weight of 40 kD, an isoelectric point of 9.0, and lacked carbohydrate or lipid components. Binding to a specific membrane protein might represent the first step of a carrier mediated uptake process. Therefore, the uptake kinetics of oleate by isolated rat heart myocytes was determined under conditions where only cellular influx and not metabolism occurred. Uptake revealed saturation kinetics and was temperature dependent which were considered as specific criteria for a facilitated transport mechanism. For evaluation whether uptake is mediated by MFABP, the effect of a monospecific antibody to this protein on cellular influx of oleate was examined. Inhibition of uptake of fatty acids but not of glucose by the antibody to MFABP indicated the physiologic significance of this protein as transmembrane carrier in the cellular uptake process of fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of partially reassembled high-density lipoprotein to isolated human small intestine epithelial cells. Effect of lipid composition

The binding of human high-density lipoprotein (HDL 3), apolipoprotein A-I (apoA-I) and recombinants of apoA-I with cholesterol and/or dimyristoylphosphatidylcholine (DMPC) to the HDL receptor on isolated human small intestine epithelial cells was studied. ApoA-I competed for 125I-labelled HDL 3 binding sites less effectively than HDL 3, and a lower amount of 125I-labelled apoA-I than 125I-HDL 3 was bound to cells. The apoA-I/DMPC recombinant competed for 125I-HDL 3 binding sites nearly as well as HDL 3, and 125I-apoA-I/DMPC recombinant bound to cells with at least the same efficiency as 125I-HDL 3. The apoA-I/DMPC/ cholesterol recombinant failed to compete for 125I-HDL 3 binding sites, and the 125I-apoA-I/DMPC/ cholesterol complex binding to cells was several-fold lower than that of other particles. All particles bound to cells with similar dissociation constants. Tetranitromethane-modified HDL 3 failed to bind to high-affinity specific binding sites and compete with 125I-HDL 3 for binding. The results obtained make it possible to assume that, while apoA-I may be a determinant of the HDL receptor, the lipid composition of the lipoprotein may affect its interaction with the receptor.

Insulin-like growth factor-I can mediate autocrine proliferation of human small cell lung cancer cell lines in vitro.

The effect of insulin-like growth factor I (IGF-I) on growth of small cell lung cancer (SCLC) cell lines was studied. Western blot analysis of whole cell lysates of cell lines NCI-H345 and NCI-N417 demonstrated the presence of a 16-kD band consistent with an IGF-I precursor molecule. Scatchard plot analysis of cell line NCI-H345 using 125I-labeled IGF-I demonstrated two high affinity specific binding sites (Kd 1.3 and 4.0 nM with maximal rate (Bmax) 200 and 500 fmol/mg protein, respectively). The exogenous addition of IGF-I, IGF-II, or insulin resulted in marked proliferation of human SCLC cells as evaluated using an in vitro growth assay. These peptides stimulated the growth of SCLC cell lines NCI-H82, NCI-H209, NCI-H345, and NCI-N417. The concentration of IGF-I producing maximal SCLC cell growth was 10-100-fold less than that of insulin or IGF-II, whereas the maximal growth stimulated by the optimal concentration of these peptides were similar. An MAb that specifically binds to the IGF-I receptor (but not to the insulin receptor) mediates a dose-dependent inhibition of cell growth in basal media as well as IGF-I, IGF-II, or insulin-supplemented media. The IGF-I receptor thus appears to be the common pathway for the mitogenic activity by IGF-I, IGF-II, and insulin for human SCLC cell lines. The demonstration of an IGF-I precursor molecule, specific IGF-I receptor binding, IGF-I-mediated growth stimulation, and inhibition of basal cell growth by an MAb to the IGF-I receptor suggests that an IGF-I-like molecule can function in vitro as an autocrine growth factor for human SCLC cell lines.

Specific opioid binding sites for dermorphin in rat brain: A radioreceptor assay using the tritiated hormone as primary ligand

Dermorphin, a heptapeptide amide isolated from amphibian skin, is the most potent of the naturally occurring opioid peptides. (3H)-dermorphin (52 Ci/mmol, 1294 GBq/mmol) was prepared by catalytic tritiation of the synthetic (2,5-iodotyrosyl 1,5)-dermorphin precursor. High affinity specific binding sites for dermorphin were labeled in rat brain membranes using tritiated dermorphin as primary ligand. The binding was saturable and time-dependent. Scatchard analysis revealed a single population of non-interacting high affinity sites (Kd = 0.86 nM). Dermorphin and the specific opiate antagonist naloxone inhibited specific (3H)-dermorphin binding in a concentration dependent manner. The displacement curves could be fit to a simple competitive model assuming only one population of binding sites, with IC 50 of 1.6 nM and 3.4 nM for dermorphin and naloxone, respectively. The use of tritiated dermorphin will be helpful to ascertain unequivocally the selectivity of dermorphin for the different opioid receptor subtypes in the central nervous system.

Distribution of neurotensin binding sites in the caudal brainstem of the rat: A light microscopic radioautographic study

Specific high-affinity neurotensin binding sites were labeled in sections of the rat caudal brainstem using a monoiodinated ligand, and their distribution was examined by light microscopic radioautography after fixation with glutaraldehyde. In the medulla, labeled binding sites were mainly concentrated within the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the external cuneate nucleus, the lateral reticular nucleus, the medial vestibular nucleus, the retrofacial nucleus, the linearis nucleus, the paragigantocellular nucleus and the nucleus raphe pallidus. Within the pons, neurotensin binding sites were detected in the reticulotegmental nucleus, the pontine nuclei, the dorsal tegmental nucleus, the laterodorsal and pedunculopontine tegmental nuclei and the nuclei raphe dorsalis and medianus. Most nuclei found here to contain high densities of neurotensin binding sites have been shown to stain intensely for acetylcholinesterase, suggesting a possible association between this enzyme and neurotensin receptors.

Binding of high density lipoproteins to cell receptors promotes translocation of cholesterol from intracellular membranes to the cell surface.

Cultured cells have on their cell surface a specific high-affinity binding site (receptor) for high density lipoproteins (HDL) which appears to promote cholesterol efflux. In this study we characterized the cellular mechanisms involved in HDL receptor-mediated transport of cholesterol from cultured human fibroblasts and bovine aortic endothelial cells. HDL3, chemically modified by tetranitromethane (TNM-HDL3), is not recognized by this receptor and was used as a control for efflux not mediated by HDL receptor binding. HDL3 and TNM-HDL3 were found to be equally effective in causing efflux of plasma membrane cholesterol radiolabeled with [3H]cholesterol. However, HDL3 was much more effective than TNM-HDL3 in causing efflux of [3H]cholesterol associated with intracellular membranes. By measuring movement of endogenously synthesized [3H]cholesterol to the plasma membrane, and into the medium, we found that HDL3 induced a rapid movement of [3H]cholesterol from a preplasma membrane compartment to the plasma membrane that preceded [3H]cholesterol efflux. This effect was not observed with TNM-HDL3. Thus, receptor binding of HDL3 appears to facilitate removal of cellular cholesterol from specific intracellular pools by initiation of translocation of intracellular cholesterol to the plasma membrane.

Binding of human monomeric type I collagen to platelets

Interaction of platelets with subendothelial collagen is important in primary hemostasis and thrombosis. Although activation of platelets by collagen polymers has been widely investigated, only insufficient data are available concerning the binding of genetically distinct collagen types in their triple helical (monomeric) form to platelets. We report on the binding of 125I-labeled human type I collagen to platelets. The binding assay was performed at 20°C in the presence of arginine in order to prevent polymerization of the collagen monomers. The binding of monomeric 125I-labeled human type I collagen is dose- and time-dependent, saturable and specific, since it is competitively inhibited by unlabeled type I collagen, but not by unlabeled human type V collagen. Scatchard analysis reveals a class of specific high affinity binding sites with a K d of 2.5 · 10 −8 M. These results suggest that platelets interact with type I collagen through specific binding sites, and that there are various different binding sites on the platelet membrane for the genetically distinct collagen types.

Protein kinase C in the nervous system of insects: effects of phorbol esters on cholinergic synapses

Specific high affinity binding sites for phorbol esters have been demonstrated in the nervous tissue of locust. The binding was reduced by the kinase C-inhibitor polymyxin B. Phorbol esters in micromolar concentrations activated protein kinase C in subcellular preparations from locust ganglia. The same concentrations of phorbol esters significantly enhanced the high affinity transport of choline and the release of acetylcholine by locust synaptosomes.

Effects of arginine vasopressin on protein phosphorylation in rat hippocampal synaptic membranes.

Our laboratory has reported previously the characteristics of specific AVP binding to rat hippocampal synaptic membranes (SPM) in the presence of Ni2+ [Costantini MG, Pearlmutter AF: J Biol Chem 259: 11739-11745, 1984]. We extended our investigation to determine the effects of Ni2+, (AVP), and AVP analogs on SPM protein phosphorylation. Ni2+ (5 mM) caused a dramatic reduction in phosphorylation of most SPM phosphoproteins. The most prominent protein which is phosphorylated in SPM has a molecular weight of 48 kilodaltons (KDa) and has been named B50 or F1; this protein shows altered phosphorylation in vitro in response to long-term potentiation in vivo as well as changes induced by exposure of SPM to ACTH (1-24), dopamine, and somatostatin. AVP and related peptides reduced phosphorylation of this pre-synaptic phosphoprotein in the following order of potency: AVP = oxytocin greater than DG-AVP greater than dDAVP greater than d(CH2)5Tyr(Me)AVP = [pGlu4,Cyt6]AVP-(4-9). Except for the pressor antagonist d(CH2)5Tyr(Me)AVP, this corresponds to their relative efficacy in displacing 3H-AVP from high-affinity specific binding sites on rat hippocampal synaptic membranes. Ni2+ did not alter the degree of inhibition caused by the peptides. When SPM were treated with AVP after the attainment of maximum 32P incorporation, AVP inhibited dephosphorylation over a 30-min period. Our results show that AVP can alter both phosphorylation and dephosphorylation of hippocampal SPM phosphoproteins in vitro; the direction of these effects depends upon experimental conditions. Since B50/F1 is known to be a substrate for protein kinase C, AVP may act by inhibition of protein kinase C activity, either directly or indirectly.

Quantitativein VivoAutoradiographic Localization of [125I_Tyr11] Somatostatin-14-and [Leu8,d-Trp22-125ITyr-125] Somatostatin-28-Binding Sites in Rat Brain*

Quantitative in vivo autoradiography was used to identify and compare the regional distribution of specific binding sites for blood-borne [125I-Tyr11]somatostatin-14 (S-14 section) and [Leu8,D-Trp22-125I-Tyr25]somatostatin-28 (S-28 section) in the rat brain. Rats were given intracardiac injections of 17 pmol S-14 section (with or without unlabeled S-14) or 17 pmol S-28 section (with or without unlabeled S-28). After whole body perfusion and fixation, brains were processed for light microscopic autoradiography of S-14 section- and S-28 section-binding sites. Of the peripheral tissues, the adrenal glands showed the highest uptake of S-14 section and S-28 section (determined by counting) and were subsequently processed for autoradiography for comparison with brain. Specific autoradiographic grains (ARG) associated with both radioligands were identified only in the circumventricular organs (CVOs). The highest ARG density associated with S-14 section was found in the area postrema, followed in decreasing order by the subfornical organ and the organum vasculosum lamina terminalis region. Median eminence (ME) contained virtually no specific S-14 section ARG. As with S-14 section, the highest ARG density of S-28 section-binding sites was also found in the area postrema, which labeled approximately equally with the two radioligands. This was followed by the ME, subfornical organ, and organum vasculosum lamina terminalis. The overall patterns of labeling of the CVOs with S-14 section and S-28 section showed significant differences, especially in the ME. Within the ME, labeled S-28 section was concentrated in a broad band throughout the external zone in a location identical to that of immunoreactive S-14. Analysis of dose-response curves obtained with 0.3-30 nmol unlabeled S-14 or S-28 revealed IC50 values for S-14 3- to 6-fold lower than those for S-28 for all labeled CVOs. With both S-14 section and S-28 section, the labelling density of the adrenal glands was double that of the area postrema. Adrenal binding of the radioligands was confined to the cells of the zona glomerulosa. We conclude: specific high affinity binding sites for S-14 section and S-28 section exist in the CVOs and the adrenal glomerulosa; and the 3- to 6-fold higher affinity of binding of S-14 to CVOs compared to S-28 together with the dissimilar patterns of labelling of the different CVOs by the two radioligands suggest the existence of separate populations of S-14 and S-28 receptors.

Calcium antagonists and their mode of action: an historical overview.

The Ca2+ antagonists are a novel group of drugs useful in management of a variety of cardiac disorders. They differ from one another in terms of their chemistry, tissue specificity and selectivity. As a group, however, they share the common property of slowing Ca2+ entry through voltage-activated, ion-selective channels. Some of them exhibit other properties, including that of interfering with Na+ transport. At least one of them, diltiazem, has an intracellular action. Specific high and low affinity binding sites have been identified for two of the major groups of Ca2+-antagonists, with the binding sites for verapamil and its derivatives being distinct from those which can be occupied by the dihydropyridines. The number (Bmax) and affinity (KD) of these binding sites changes under certain pathological conditions--including a reduction in ischaemia and in spontaneous hypertension, an increase in the latter, at present, only demonstrated for the dihydropyridine binding sites. The sensitivity of a particular tissue to these drugs will depend upon a number of factors including the number of binding sites that are present, the contribution made by the Ca2+ entering through the voltage-activated channels to the functioning of the tissue, and properties which are peculiar to a particular type of Ca2+ antagonist, for example, whether, as in the case of verapamil, they exhibit use-dependence.

Calcium-induced membrane metabolic alterations modify the sex steriods binding into dog brain synaptosomal plasma membranes

1. 1. The binding of 45Ca 2+ into synaptosomal plasma membranes (SPM) of dog brain follows a sigmoid path. In graphical analysis of this binding the mean Hill coefficient ( h) was 1.64 ± 0.09 ( r 2 = 0.96 ± 0.02). Binding of Ca 2+ into SPM was saturable, with an apparent binding constant of 1.2 ± 0.1 μM. At saturation, such calcium specific binding sites corresponded to 11.2 ± 0.9 nmol/mg SPM protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperatively in the binding of calcium into SPM of at least one class of high affinity specific binding sites. 2. 2. [ 14C]estradiol, [ 14C]estrone and [ 14C]progesterone, when incubated with SPM up to a concentration of 10 μM for 2 hr at 37°C, bind into SPM at nmolar concentrations. Ca 2+ ions up to 5 mM considerably increase steroids binding into SPM. This effect of calcium as concentration-dependent, reached saturation at approx 4–5 mM. Once calcium has promoted steroids binding, the subsequent addition of 25 mM EGTA failed to displace bound steroids. 3. 3. Molecular interactions between calcium and SPM was assessed by measuring the steady-state fluorescence polarization (P) of 1,6-diphenyl-1,3,5-hexatriene (DPH), and by estimating the production of malondialdehyde (MDA) during 2 hr incubation of Ca 2+ (5 mM) with SPM at 37°C. The effect of Ca 2+ on the SPM structure was to increase both the rigidity of the membrane and the MDA production. 4. 4. Chelation of Ca 2+ (5 mM) with EGTA (25 mM) did not reverse the increase in the rigidity owing to metabolic alterations of SPM lipids (e.g. production of MDA). 5. 5. Arrhenius plots of 5′-nucleotidase activity exhibited a break point at 23.2°C for untreated (control) SPM, which was elevated to 29.5°C in the SPM treated with calcium, consistent with a general increase in membrane rigidity. 6. 6. These studies provide a basis for postulating a mechanism of calcium ions induce increase of sex steroids binding in membranes of brain cells by increasing the membrane rigidity.

Antiestrogen binding within different pituitary cell populations. Comparison with androgen and estrogen receptors

Gonadotrope-enriched populations were prepared from 42-day old male rats by centrifugal elutriation. They contained 4.8 ± 0.7% of the cells, 51 ± 10% of the LH and less than 3% of the PRL ( n = 4). Gonadotrope-depleted fractions were also obtained that contained most of PRL cells. Specific antiestrogen binding sites (AEBS) were quantitied in these populations after destruction of estrogen receptor. Results showed the presence of a distinct, specific high affinity binding site for antiestrogen in dispersed pituitary cells and in enriched fractions. However, AEBS are not specific of a pituitary cell type. Thus, AEBS appear different from estrogen receptors in pituitary gland: (1) by the thermal stability of AEBS, (2) by the localization of AEBS in pariculate material, (3) by the uniform distribution of AEBS in different populations which differ markedly for E 2 binding sites. Whereas the ratio of binding AE/E 2 averaged 11.4 in the initial cell suspension it reached only 2.9 in the gonadotropes. The dissociation constants for AEBS were in the same range (1.16—2.27-10 −9M) for the different populations.

Binding of high density lipoprotein to cultured fibroblasts after chemical alteration of apoprotein amino acid residues.

Cultured extrahepatic cells possess a specific high affinity binding site (receptor) for high density lipoprotein (HDL) that is induced by cholesterol delivery to cells. To characterize the binding recognition site(s) on HDL, the ability of HDL to interact with cultured human fibroblasts was assayed after chemical alteration of specific apoprotein amino acid residues. Reduction and alkylation, acetylation, and cyclohexanedione treatment of HDL3 had little or no effect on its cellular binding. Treatment of HDL3 with tetranitromethane (TNM), however, caused a large dose-dependent decrease in binding, with maximum inhibition at 3 mM. Amino acid analysis of the TNM-treated particles showed specific alteration of tyrosine residues, but sodium dodecyl sulfate-gel electrophoresis demonstrated apoprotein cross-linking coincident with decreased binding. These results suggest that modification of HDL tyrosine residues and/or cross-linking of HDL apoproteins alters the ligand site recognized by the HDL receptor. Gradient gel electrophoresis, molecular sieve chromatography, and electron microscopy showed only minor changes in size distribution and shape of HDL3 particles after treatment with 3 mM TNM, but at higher TNM concentrations, coalescence and aggregation of particles was evident. Treatment of HDL3 with 3 mM TNM affected neither its promotion of the low affinity (receptor-independent) cholesterol efflux from cells nor its ability to accept cholesterol from an albumin suspension, yet promotion of high affinity (receptor-dependent) cholesterol efflux from cells was abolished. The finding that TNM treatment of HDL3 decreases both its receptor binding and its promotion of cholesterol efflux from cells without substantial alteration of its physical properties supports the hypothesis that the HDL receptor functions to facilitate cholesterol transport from cells.