High-density lipoprotein binding to guinea-pig hepatic membranes. Comparison of guinea-pig and human ligands

Human and guinea-pig apo-E-free HDL were incubated with isolated guinea-pig hepatic membranes at 4 and 37 degrees C to determine the effects of temperature and heterologous systems on the equilibrium parameters of HDL binding. Receptor mediated HDL binding was highest at 37 degrees C for both lipoproteins. At 4 degrees C, a higher affinity constant (Kd) was observed when guinea-pig HDL was the ligand relative to human HDL; in contrast, both HDL preparations had similar Kd values at 37 degrees C. Calculated binding and receptor number (Bmax) were higher at both temperatures when guinea-pig HDL was the ligand. These results demonstrate a significant species difference in HDL binding to hepatic membrane which is partially temperature-dependent.     

Identification of estrogen receptors in granulosa cells of immature rats

Nuclear and cytoplasmic binding sites for estradiol (E2-17 beta) in granulosa cells of immature rats were characterized. These binding sites for estrogen were high affinity, low capacity with an affinity constant (Kd) of 1.9 X 10(-10)M (binding capacity, Ro = 80 pM) for nuclear sites and a Kd = 3.5 X 10(-10) M (Ro = 45 pM) for cytosol sites. Binding was specific for biologically active estrogens. The estrogen receptor in granulosa cells is a protein and heat-labile as treatment with protease or pre-incubation at 37 degrees C for 1 h significantly diminished binding. RNase and DNase had no effect on estrogen binding. Sedimentation coefficients for nuclear and cytosol binding components were 5S and 8S respectively, similar to values obtained with uteri. Finally, translocation was demonstrated after a s.c. injection of E2-17 beta. Forty-five minutes post-injection, cytosol binding sites for estradiol were depleted concomitant with accumulation of nuclear binding sites. We concluded that granulosa cells of immature rats have binding sites specific for estradiol which have characteristics similar to the classical estrogen receptor in uteri.     

Temperature dependence and GABA modulation of [3H]triazolam binding in the rat brain

The hypnotic triazolam (TZ), a triazolobenzodiazepine displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Our major objectives were the direct measurement of the temperature dependence and the gamma-aminobutyric acid (GABA) effect of [3H]TZ binding in the rat brain. Saturation studies showed a shift to lower affinity with increasing temperatures (Kd = 0.27 +/- 08 nM at 0 degree C; Kd = 1.96 +/- 0.85 nM at 37 degrees C) while the Bmax values remained unchanged (1220 +/- 176 fmoles/mg protein at 0 degree C and 1160 +/- 383 fmoles/mg protein at 37 degrees C). Saturation studies of [3H]TZ binding in the presence or absence of GABA (100 microM) showed a GABA-shift. At 0 degrees C the Kd values were (Kd = 0.24 +/- 0.03 nM/-GABA; Kd = 0.16 +/- 0.04/+GABA) and at 37 degrees C the Kd values were (Kd = 1.84 +/- 0.44 nM/-GABA; Kd = 0.95 +/- 0.29 nM/+GABA). In contrast to reported literature, our findings show that TZ interacts with benzodiazepine receptors with a temperature dependence and GABA-shift consistent with predicted behavior for benzodiazepine agonists.     

Estradiol binding components in intestinal mucosa of female rats.

The presence of estrogen binding components (EBC) in intestinal mucosa of female rats was investigated by competitive-binding assay using radiolabelled and nonlabelled estradiol 17 beta (E2). EBC were found exclusively in the nuclear fraction and were absent from the cytosolic and from the microsomal fractions. Two types of nuclear EBC with different binding characteristics and capacities were found: Kd1 = 4.8 +/- 0.8 nM, n1 = 18.4 +/- 4.2 fmol/mg protein (n1 = 83.4 +/- 12.5 fmol/mg DNA) and Kd2 = 31.1 +/- 6.8 nM, n2 = 91.1 +/- 18.5 fmol/mg protein (412.7 +/- 80.0 fmol/mg DNA). Type 1 component showed slightly greater affinity for estrogens as compared to progesterone and dexamethasone whereas type 2 component bound other competitors with even greater affinity than E2.     

Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea (+/- 0.4 M KCl) buffers and control buffers (+/- 0.4 M KCl) by chromatography through small columns of Sephadex G-25 or by dialysis at 0-6 degrees C. Equilibrium dissociation constants (Kd) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17 beta-[3H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol (Kd = 0.36 +/- 0.09 nM, n = 14) which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity (Kd = 3.45 +/- 0.86 nM, n = 6) to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. However, KCl did help prevent the reduction in binding capacity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor binding of selectively labeled (Tyr-10) and (Tyr-13)-mono-125I-glucagons and competition by homologous 127I-labeled isomers

Two monoiodinated derivatives of glucagon were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The purified (Tyr-10) and (Tyr-13)-mono-125I-labeled glucagon isomers were characterized and studied with respect to their binding to the receptors of isolated intact rat hepatocytes. The extent of steady-state binding to cellular receptor sites differed for the two labeled glucagon tracers at 37 degrees C as well as at 15 degrees C with (Tyr-10)-mono-125I-glucagon displaying higher receptor binding. The apparent equilibrium constants, Kd,app at 37 degrees C are 3.6 +/- 0.4 nM (mean +/- S.E. of three independent experiments) for the tyrosine-13-labeled tracer and 5.9 +/- 0.6 nM for the tyrosine-10-labeled glucagon with native glucagon as competitor. Since the observed Kd in the competition assay is a function of the true Kd values of the monoiodinated radioactive glucagon isomers and native glucagon, the dissociation constants were also measured with chemically identical tracer and competitor. Under these conditions, we obtained Kd values of 1.3 +/- 0.2 nM for the tyrosine-10-labeled analog and 2.0 +/- 0.2 nM for the tyrosine-13-labeled glucagon isomers confirming the higher receptor binding affinity of (Try-10)-mono-125I-glucagon. All competition curves fit the mathematical expression for a model of non-cooperative binding to a single class of receptors.     

Identification of an alpha 2-adrenoceptor in human coronary arteries by radioligand binding assay.

A single high affinity binding site for an alpha 2-adrenoceptor in human coronary arteries was identified by radioligand binding assay. Human coronary arteries were obtained at autopsy within 6 hours of death. A crude membrane solution was incubated with (3H)-rauwolscine at 25 degrees C for 30 min. The binding of (3H)-rauwolscine was rapidly saturable and reversible. Kd was 1.2 +/- 0.2 (SE) nM and Bmax 22 +/- 3 fmol/mg protein. This is the first study which has shown the presence of an alpha 2-adrenoceptor in human coronary arteries using a radioligand binding assay method.     

Multiple affinity states of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Opiate agonist association rate is a function of receptor occupancy

The existence of multiple affinity states for the opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells has been demonstrated by competition binding studies with tritiated diprenorphine and [D-Ala2, D-Leu5]enkephalin (DADLE). In the presence of 10 mM Mg2+, all receptors exist in a high affinity state with Kd = 1.88 +/- 0.16 nM. Addition of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) decreased the affinity of DADLE to Kd = 8.08 +/- 0.93 nM. However, in the presence of 100 mM Na+, which is required for opiate inhibition of adenylate cyclase activity, analysis of competition binding data revealed three sites: the first, consisting of 17.5% of total receptor population has a Kd = 0.38 +/- 0.18 nM; the second, 50.6% of the population, has a Kd = 6.8 +/- 2.2 nM; and the third, 31.9% of the population, has a Kd of 410 +/- 110 nM. Thus, in the presence of sodium, a high affinity complex between receptor (R), GTP binding component (Ni), and ligand (L) was formed which was different from that formed in the absence of sodium. These multiple affinity states of receptor in the hybrid cells are agonist-specific, and the percentage of total opiate receptor in high affinity state is relatively constant in various concentrations of Na+. Multiple affinity states of opiate receptor can be demonstrated further by Scatchard analysis of saturation binding studies with [3H]DADLE. In the presence of Mg2+, or Gpp(NH)p, analysis of [3H]DADLE binding demonstrates that opiate receptor can exist in a single affinity state, with apparent Kd values of [3H]DADLE in 10 mM Mg2+ = 1.75 +/- 0.28 nM and in 10 microM Gpp(NH)p = 0.85 +/- 0.12 nM. There is a reduction of Bmax value from 0.19 +/- 0.02 nM in the presence of Mg2+ to 0.14 +/- 0.03 nM in the presence of Gpp(NH)p. In the presence of 100 mM Na+, Scatchard analysis of saturation binding of [3H]DADLE reveals nonlinear plots; two-site analysis of the curves yields Kd = 0.43 +/- 0.09 and 7.9 +/- 3.2 nM. These Kd values are analogous to that obtained with competition binding studies. Again, this conversion of single site binding Scatchard plots to multiple sites binding plots in the presence of Na+ is restricted to 3H-agonist binding only.(ABSTRACT TRUNCATED AT 400 WORDS)     

High affinity angiotensin II receptors in myocardial sarcolemmal membranes. Characterization of receptors and covalent linkage of 125I-angiotensin II to a membrane component of 116,000 daltons

High affinity receptors for angiotensin II have been identified on purified cardiac sarcolemmal membranes. Equilibrium binding studies were performed with 125I-labeled angiotensin II and purified sarcolemmal vesicles from calf ventricle. The curvilinear Scatchard plots were evaluated by nonlinear regression analysis using a two-site model which identified a high affinity site Kd1 = 1.08 +/- 0.3 nM and N1 = 52 +/- 10 fmol/mg of protein and a low affinity site Kd2 = 52 +/- 16 nM and N2 = 988 +/- 170 fmol/mg of protein. Monovalent and divalent cations inhibited the binding of 125I-angiotensin II by 50%. The affinity of angiotensin II analogs for the receptor was determined using competitive binding assays; sarcosine, leucine-angiotensin II (Sar,Leu-angiotensin II), Kd = 0.53 nM; angiotensin II, Kd = 2.5 nM; des-aspartic acid-angiotensin II, Kd = 4.81 nM; angiotensin I, Kd = 77.6 nM. There is a positive correlation between potency in inducing positive inotropic response in myocardial preparations reported by others and potency for the hormone receptor observed in the binding assays. Pseudo-Hill plots of the binding data showed that agonists display biphasic binding with Hill numbers around 0.65 while antagonists recognized a single class of high affinity receptors with Hill numbers close to unity. These data were confirmed using 125I-Sar,Leu-angiotensin II in equilibrium binding studies which showed that this antagonist bound to a single class of receptor sites; Kd = 0.42 +/- 0.04 nM and N = 1050 +/- 110 fmol/mg of protein. Competition-binding experiments with this 125I-peptide yielded monophasic curves with Hill numbers close to unity for both agonists and antagonists. Membrane-bound 125I-angiotensin II was covalently linked to its receptor by the use of bifunctional cross-linking reagents such as dithiobis(succinimidyl propionate) and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. Analysis of the membranes showed the labeling of a component with an apparent Mr = 116,000. The affinity labeled species showed characteristics expected of a functional component of the high affinity receptor. The affinity labeling of this membrane component was inhibited by nanomolar angiotensin II or Sar,Leu-angiotensin II. Together these data indicate that high affinity receptors exist for angiotensin II that most likely mediate the positive inotropic effects of this hormone on myocardial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of antigenic peptides with MHC class I molecules on living cells studied by photoaffinity labeling.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of antigenic peptides with murine MHC class I molecules on living cells. Photoreactive derivatives were prepared by N-terminal amidation with iodo, 4-azido salicylic acid of the Kd restricted Plasmodium berghei circumsporozoite (P.b. CS) peptide 253-260 (YIPSAEKI) and the Db-restricted Adenovirus 5 early region 1A (Ad5 E1A) peptide 234-243 (SGPSNTPPEI). As assessed in functional competition experiments, both peptide derivatives retained the specific binding activity of the parental peptides for Kd or Dd, respectively. The P.b. CS photoprobe specifically labeled Kd molecules on P815 (H-2d) cells, but failed to label RMA (H-2b) cells. Conversely, the Ad5 E1A photoprobe specifically labeled Db molecules on RMA cells, but failed to label P815 cells. When the two photoprobes were tested on a panel of Con A-activated spleen cells expressing 10 different H-2 haplotypes, significant photoaffinity labeling was observed only on H-2d cells with the P.b. CS photoprobe and on H-2b cells with the Ad5 E1A photoprobe. Labeling of cell-associated Kd or Db molecules with the photoprobes was specifically inhibited by antigenic peptides known to be presented by the same class I molecule. Photoaffinity labeling of Kd with the P.b. CS photoprobe was used to study the dynamics of peptide binding on living P815 cells. Binding increased steadily with the incubation period (up to 8 h) at 37 degrees C and at ambient temperature, but was greatly reduced (greater than 95%) at 0 to 4 degrees C or in the presence of ATP synthesis inhibitors. The magnitude of the labeling was twofold higher at room temperature than at 37 degrees C. In contrast, binding to isolated Kd molecules in solution rapidly reached maximal binding, particularly at 37 degrees C. Dissociation of the photoprobe from either cell-associated or soluble Kd molecules was similar, with a half time of approximately 1 h at 37 degrees C, whereas the complexes were long-lived at 4 degrees C in both instances.     

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple classes of prostaglandin F2 alpha binding sites in subpopulations of ovine luteal cells

A cryostorage procedure was developed to provide ovine luteal cells throughout the period of seasonal anestrus. Corpora lutea obtained from midluteal phase, superovulated ewes were dispersed enzymatically. Some dispersed cells were fractionated into subpopulations by elutriation. Dimethylsulfoxide (7.5% final concentration) in Hanks' buffered saline was added to cells at 4 degrees C, and dispersed cell preparations were frozen in a programmable cell freezer and stored at -196 degrees C. After recovery from cryopreservation, cell viability and prostaglandin F2 alpha (PGF2 alpha) binding characteristics of thawed cells were not different from those of corresponding fresh cells. Additionally, thawed cells retained the capacity to attach to culture dishes and retained responsiveness of progesterone secretion to prostaglandin E2 (PGE2) and ovine luteinizing hormone (LH), although rates of progesterone secretion were attenuated in thawed compared with fresh cells. The cryopreservation procedure will prove useful to relieve constraints in utilization of ovine luteal cells arising from reproductive seasonality in sheep. Cells retrieved from cryostorage were evaluated by studying PGF2 alpha binding characteristics. From saturation analyses (increasing amounts of radiolabeled PGF2 alpha) of PGF2 alpha binding to unfractionated cells, we detected a single class of high affinity binding sites (Kd = 17.4 +/- 2.3 nM) in addition to the nonspecific binding component. Using displacement analyses (constant radiolabeled PGF2 alpha and increasing amounts of unlabeled PGF2 alpha) and unfractionated cells, we detected additional binding sites of lower affinity (Kd = 409 +/- 166 nM) as well as the nonspecific binding component. Small luteal cells obtained by elutriation, which were essentially devoid of large cell contamination, had only low affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms.

When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [( 3H]arachidonate mobilization and thromboxane synthesis) and secretion [( 14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding via mutually independent mechanisms.     

Presence of two types of estrogen binding sites in mouse testis

The characteristics of cytosol estrogen binding sites in BALB/c mouse testis were investigated. The cytosol prepared from the whole testis contained two classes of the specific estrogen binding sites by Scatchard and Rosenthal plot analyses. The first binding site (first binder) had high affinity for 17 beta-estradiol (E2; Kd = 4.9 X 10(-9) M) and binding specificity as observed in the typical estrogen receptor. The second binding site (second binder) had lower affinity for E2 (Kd = 4.8 X 10(-8) M) and the binding was inhibited less vividly by diethylstilbestrol (DES) and antiestrogens in comparison with that for the first binder. Postlabeled sucrose density gradient analysis in a low salt medium revealed that the major radioactive peak of the first binder appeared at 7S region, while that of the second binder sedimented at 4S region. The 7S component showed an appreciable binding to the nuclei, while the 4S component did not show a significant binding ability to the nuclei. Much higher concentrations of the first and the second binders were found in Leydig cells preparations. These results demonstrate the presence of two types of the specific estrogen binding sites in the mouse testis especially in Leydig cells.     

11 beta-chloromethyl-[3H]estradiol-17 beta: a very high affinity, reversible ligand for the estrogen receptor

It has been suggested that binding of 11 beta-chloromethyl estradiol (11 beta-CME2) to the estrogen receptor is irreversible, since its complex with receptor fails to undergo exchange with estradiol (E2). To investigate this behavior directly, 11 beta-CME2 was prepared in high specific activity, tritium-labeled form: The binding of [3H]11 beta-CME2 to the estrogen receptor from lamb and rat uterus and MCF-7 human breast cancer cells was shown to be fully reversible; the 11 beta-CME2 complex with receptor, as well as that of a structural analog 11 beta-ethyl estradiol, however, do not dissociate or exchange with [3H]E2 over a 22 h period at 25 degrees C. By competitive or direct binding assays, the affinity of 11 beta-CME2 for the estrogen receptor can be estimated to be as much as 10- to 30-fold higher than that of E2. The complexes of estrogen receptor from MCF-7 cells with [3H]11 beta-CME2 and [3H]E2 show identical velocity sedimentation profiles on sucrose gradients, under conditions when the receptor is either a monomer of a dimer. Because of its very high affinity and unusual dissociation kinetics, [3H]11 beta-CME2 should be a very useful ligand for studies of estrogen receptor dynamics and in the assay of estrogen receptor concentrations in tumors and tissues.     

The interaction of vasoactive intestinal peptide (VIP) with isolated bovine thyroid plasma membranes

The binding of vasoactive intestinal peptide (VIP) and stimulation of adenylate cyclase were studied in bovine thyroid plasma membranes. The binding depended on time, temperature and was saturable and specific. Binding studies suggested the presence of two classes of binding sites: a class with high affinity (Kd = 13 nM) and low capacity (6411 sites/pg), and a class with low affinity (Kd = 480 nm) and high capacity (105,300 sites/pg) at 15 degrees C. Secretin, glucagon, insulin and somatostatin did not displace the tracer from the membranes. VIP stimulated cyclic AMP production. Maximal cyclic AMP production (2-fold above basal values) was observed with 100 nM VIP and half-maximal response was obtained at 5 nM VIP at 15 degrees C.     

Binding of estrogen-3-sulfates to stallion plasma and equine serum albumin.

The binding of estrone-3-sulfate (E1-3-S) and estradiol-3-sulfate (E2-3-S) to adult stallion plasma was determined and compared with the binding to equine serum albumin (ESA). On the ESA molecule, two binding sites for E1-3-S with an association constant of 1.3 x 10(5) M-1 and several sites of weaker affinity were found; the data for E2-3-S showed the existence of four binding sites of moderate affinity (1 x 10(5) M-1) and several sites of weaker affinity. The removal of albumin from the stallion plasma resulted in the absence of binding of E1-3-S or E2-3-S, whereas the removal of glycoproteins resulted in binding parameters similar to those obtained with whole plasma. These results indicate that ESA is the only estrogen sulfate binder in horse plasma. Under physiological conditions, 95% of E1-3-S was bound to ESA.     

Evidence for two sites on rat liver plasma membranes which interact with high density lipoprotein.

There is little dispute that high density lipoprotein (HDL) binds to cells, however, the nature of the interaction is not fully understood. We now present evidence for a new binding site of higher affinity but lower capacity than the sites previously described in the literature. This new site is characterized by high affinity/low capacity for HDL binding (Kd = 0.94 microgram/ml, Bmax = 36 ng/mg), while the low affinity site (Kd = 36 micrograms/ml, Bmax approximately 700 ng/mg) appears to be consistent with the literature values for the interaction of HDL with cells and isolated membranes. Proteolysis of HDL with trypsin abolished its interaction with the high affinity site, suggesting an apolipoprotein requirement, while having no effect on binding to the lower affinity site. Kinetic rates of association/dissociation were determined in order to further characterize the high affinity site. At a concentration which favored the binding of HDL with the high affinity site (1 microgram/ml, 37 degrees C), the time course of association of HDL with rat liver plasma membranes, displayed a biphasic pattern, requiring 6-8 h to reach the level of binding predicted from the saturation studies. The second phase was highly sensitive to temperature, being considerably slower at 24 degrees C and totally abolished at 0 degrees C. A kinetic Kd, derived from the measured association and dissociation rate constants (Kd = 0.31 microgram/ml), was found to be of a similar magnitude to the Kd calculated for the high affinity site by Scatchard analysis (Kd = 0.94 microgram/ml). In summary, the high affinity site on rat liver plasma membranes displays an apoprotein requirement and kinetic parameters, consistent with a ligand-receptor interaction.