Effect of cell density on thrombin binding to a specific site on bovine vascular endothelial cells

We studied thrombin binding to proliferating and confluent endothelial cells derived from bovine vascular endothelium. [125]thrombin was incubated with nonconfluent or confluent endothelial cells and both the total amount bound and the amount linked in a 77,000-dalton thrombin- cell complex were determined. Approximately 230,000 molecules of thrombin bound per cell in nonconfluent cultures compared to 12,800 molecules per cell in confluent cultures. Approximately 67,7000 thrombin molecules were bound in an apparently covalent complex, Mr = 77,000, with each cell in sparse cultures, whereas only 4,600 thrombin molecules per cell were bound in this complex with confluent cultures. Similar studies with [125I]thrombin and endothelial cells derived from bovine cornea revealed no difference either in the total amount of thrombin bound or in the amount bound in the 77,000-dalton complex using sparse or confluent cultures. When confluent vascular endothelial cultures were wounded, additional cellular binding sites for the 77,000- dalton complex with thrombin appeared within 24 h. A 237% increase in the amount of thrombin bound to these sites was induced by a wound which resulted in a 20% decrease in cell number in the monolayer. There was no significant increase in thrombin binding to other cellular sites at 24 h. These experiments provide evidence that the first change in thrombin binding after injury is an increase in the cellular sites involved in the 77,000-dalton complex, and suggest that thrombin binding to endothelial cells may be important in the vascular response to injury.     

Binding of thrombin to human platelet plasma membranes

Binding of ²⁵I-labeled thrombin to isolated human platelet plasma membranes was studied. Two classes of sites, one with high and one with low affinity for thrombin, were demonstrated. The apparent dissociation constants for the high and low affinity sites were 3.2 and 600 nM, respectively, similar to values obtained with intact platelets. Maximum binding was within 10 s, the shortest time measured, and then decreased with time to a constant level of binding within 45 s. When th equilibrium was perturbed by dilution, the system re-equilibrated with less thrombin bound than in a control that was diluted before mixing thrombin and membranes. Neither the time-dependent decrease nor the dilution effect were observed with phenylmethylsulfonyl-¹²⁵I-labelled thrombin, an irreversibly inhibited thrombin, suggesting that these phenomena may involve a thrombin-catalyzed modification of the membranes leading to decreased binding.     

Characterization of alpha 2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

[3H]Yohimbine, a potent alpha 2-adrenergic antagonist, was used to label the alpha-adrenergic receptors in membranes isolated from human platelets. Binding of [3H]yohimbine to platelet membranes appears to have all the characteristics of binding to alpha-adrenergic receptors. Binding reached a steady state in 2-3 min at 37 degrees C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10(-5) M; t1/2 = 2.37 min). [3H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [3H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (-) isomer was 11-times more potent that the (+) isomer. Catecholamine agonists competed for the occupancy of the [3H]yohimbine-binding sites with an order of potency: clonidine greater than (-)-epinephrine greater than (-)-norepinephrine much greater than (-)-isoproterenol. The potent alpha-adrenergic antagonist, phentolamine, competed for the sites whereas the beta-antagonist, (+/-)-propranolol, was very weak inhibitor. 0.1 mM GTP reduced the binding affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonin competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [3H]yohimbine binding. These results suggest that [3H]yohimbine binding to hunan platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label alpha 2-adrenergic receptors.     

Thrombin binds to a high-affinity approximately 900 000-dalton site on human platelets

The functional sizes of the binding sites for thrombin on human platelets and isolated membranes have been determined by the technique of radiation inactivation: similar results were obtained. Independent studies using different radiation doses (0, 3, and 48 Mrad) and different thrombin concentrations (10(-10), 10(-8), and 10(-6) M) confirmed the presence of three binding sites with functional sizes of 900 000, 30 000, and 4000 daltons. The binding site of lowest apparent size (4000 daltons) probably corresponds to what has been termed nonspecific binding since its dissociation constant (2900 nM) is well outside the physiological range. The site of intermediate size (30 000 daltons) is also probably not involved in platelet activation since its dissociation constant (11 nM) is also beyond the concentration range required for activation, although it may be involved in other aspects of platelet-thrombin interaction. The sites with the largest functional size are probably important in platelet function since their dissociation constant (0.3 nM) is in the range required for platelet activation. The functional size of these sites (900 000 daltons) suggests that the high-affinity site for thrombin binding to platelets may involve a multimolecular complex of membrane components.     

Interaction of alpha 2-macroglobulin-bound thrombin with hirudin

The human thrombin bound to alpha 2-macroglobulin (alpha 2 M) in a 1:1 stoichiometry is still able to interact with one of its specific inhibitors, hirudin. The dissociation constant of the complex hirudin--alpha 2M-bound thrombin is 1 X 10(-7) M, whatever the mode of thrombin binding, covalent or non-covalent.     

Binding of FITC-labelled alpha-thrombin by peritoneal mast cells]

The interaction of bovine alpha-thrombin with peritoneal mast cells was studied using FITC-labeled enzyme. Thrombin was modified with FITC in the presence of heparin and was separated from heparin and free FITC by gel-filtration at HPLC yielding FITC-labeled alpha-thrombin with intact additional recognition binding site for high molecular substrates and cell receptors. Equilibrium studies have shown that the binding of thrombin to peritoneal mast cells is active independent, rapid, specific, saturable and reversible. Equilibrium between bound and free thrombin is attained within I min and Scatchard analysis indicates a population of approximately 54 x 10(3) sites/cell with a dissociation constant of 1.3 x 10(-9) M. FITC-labeled alpha-thrombin binds to peritoneal mast cells in a temperature-dependent manner with optimum at 37 degrees C. These results indicate that FITC-labeled alpha-thrombin binds to peritoneal mast cells with high affinity.     

Binding of thrombin to cultured human fibroblasts: evidence for receptor modulation

Previous work from this laboratory has indicated that thrombin's influence on cell growth can be negative as well as positive. Addition of enzyme to actively growing or confluent cultures of human skin fibroblasts produced growth stimulation, whereas cultures receiving thrombin at the time of subculture displayed inhibited DNA synthesis and mitosis. The specific binding of [125I]thrombin to cells under stimulatory and inhibitory conditions has been studied. Fibroblasts receiving enzyme at subculture bound about two times more [125I]thrombin than those processed in the same way several hours later. The apparent dissociation constant for both groups was approximately 1.5 x 10(-8) M. In each case binding was saturable, although cells receiving enzyme at subculture showed a much higher rate of binding. Experiments were conducted in which enzyme was added to cells at various times after subculture. It was found that the ability of these fibroblasts to specifically bind [125I]thrombin decreased progressively over a 2-h period after subculture and then remained constant for at least 24 h. Evidence is also presented indicating that the binding of [125I]thrombin in both experimental groups was inversely dependent upon the culture density. The biological effects of elevated thrombin binding in cells receiving enzyme at subculture were examined. It was found that inhibited DNA synthesis and altered cellular morphology were directly to this parameter. This study suggests that fibroblasts may possess cryptic thrombin receptors that become exposed during subculture or after injury in vivo. These possibilities and the relationship of cell shape to the availability of thrombin receptors are discussed.     

The adsorption of prothrombin to phospholipid monolayers quantitated by ellipsometry

We investigated by means of an automated ellipsometer the calcium-dependent binding of prothrombin from a buffer solution to monolayers of dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidylcholine (DOPC) deposited on chromium slides. This technique allows direct measurements of bound and free protein concentrations and is not hampered by calcium-induced aggregation of vesicles. For pure DOPS a dominant class of binding sites exists with a dissociation constant, Kd = (6 +/- 2) X 10(-10) M (mean +/- S.D.) and maximal binding of prothrombin, gamma max = 0.26 +/- 0.03 micrograms/cm2. Incorporation of a small fraction of DOPC in the monolayer causes a large decrease in the binding affinity with a pronounced biphasic behavior of the binding curve. For monolayers consisting of 20% DOPS and 80% DOPC the binding curve becomes monophasic with Kd = (1.6 +/- 0.6) X 10(-7) M and gamma max = 0.22 +/- 0.03 micrograms/cm2. The procoagulant activity of the monolayers was tested by measuring the generation of thrombin after addition of prothrombin and activated coagulation factors X and V. The thrombin-generating capacity of monolayers and single-bilayer vesicles is comparable but is apparently diffusion limited in the monolayer system. The calcium-dependent formation of stacked multilayers according to the Blodgett technique appeared to be strongly influenced by the DOPS/DOPC ratio in the phospholipid monolayer. From these results it is concluded that for pure DOPS monolayers high-affinity prothrombin-phospholipid and phospholipid-phospholipid interactions exist which are radically disturbed when the monolayer contains more than 20-30% of DOPC.     

Thyrotropin receptors in normal human thyroid. Nonclassical binding kinetics not explained by the negative cooperativity model

Saturation analysis of equilibrium binding of iodinated thyrotropin (125I-TSH) to normal human thyroid preparations yielded linear Scatchard plots under non-physiological conditions of pH 6.0 or 20 mM Tris/acetate buffer, pH 7.4. The apparent equilibrium dissociation constant of this binding was approximately 10(-8) M. By contrast, nonlinear plots were obtained under standard conditions of pH 7.4 and 40 mM Tris/acetate buffer. Resolution of the components of these curves by computer analysis revealed the presence of at least two classes of binding sites, one of which is of a low capacity and high affinity (approximately 10(-10) M) consistent with receptor binding. The other component is of a high capacity and lower affinity. Binding to non-target tissues of muscle, parathyroid, mammary carcinoma, and placenta was only demonstrable at pH 6.0 or in 20 mM Tris/acetate buffer, pH 7.4, yielding linear Scatchard plots with similar binding affinity (approximately 10(-8)M) to normal thyroid but much reduced capacity. Preincubation of thyroid tissue at 50 degrees C resulted in an apparent selective loss of the high affinity component of binding measured under standard conditions. Kinetic experiments on the dissociation of bound 125I-TSH were undertaken to determine whether the non-linearity of Scatchard plots was due to two or more classes of binding sites or negative cooperativity. It was found that the experimental determinant that is presently ascribed to a negative cooperativity phenomenon regulating receptor affinity (i.e. an enhanced dilution-induced dissociation rate in the presence of excess native hormone), although apparently hormone-specific, was demonstrated under nonphysiological binding conditions and in non-target tissue. Significantly, the phenomenon was found under conditions of pH 6.0 or 20 mM Tris where a linear Scatchard plot was obtained. The evidence thus suggests that 125I-TSH binds to heterogeneous binding sites (of which the high affinity is probably the receptor for TSH) and that the enhanced dilution-induced dissociation of bound hormone by native hormone for this system, is only a characteristic of the low affinity binding site (maybe gangliosides).     

Dissociation of thrombin from platelets by hirudin. Evidence for receptor processing.

Hirudin inhibited the binding of human 125I-alpha-thrombin to the saturable, but not the nonsaturable, sites on washed human platelets. When hirudin was added to a thrombin-platelet mixture, it caused a biphasic dissociation of bound thrombin. A partial dissociation was too rapid to measure and was followed by complete dissociation with a first order rate constant of about 10(-2) s-1. The fraction of bound thrombin in the more slowly dissociable form increased from essentially none after a 5-s preincubation of thrombin and platelets to as much as 75% of saturable binding after a 4-min preincubation. Transition to the slowly dissociable state was not accompanied by an increase in the amount bound and was not observed with active site serine-derivatized thrombin. This is the first evidence with intact platelets of a binding characteristic that depends, as does platelet stimulation, on catalytically active thrombin, suggesting that it may represent physiologically significant receptor processing.     

Isoelectric heterogeneity of human uterine estrogen binding proteins

Upon Isoelectric Focusing (IEF) of premenopausal uterine myometrial cytosol, specific binding of estradiol (E2) can be shown at elution pH's (EpH) of 4.0-4.4, 5.0-5.2, 5.8-6.2 and 7.5-8.0. Pre-adsorption of premenopausal uterine cytosol by Concanavalin A Sepharose (Con-A) or precipitation with 30% ammonium sulfate results in loss of estradiol binding at EpH's 4.4 and 5.0. The estradiol binding sites that bind to Con-A are present in plasma and have been shown to be Sex Hormone Binding Globulin (EpH = 5.0) and Estrogen Binding Protein (EpH = 4.4). After Con-A adsorption premenopausal cytosol preincubated with 2 nM 3HE2 reveals a single peak on IEF at EpH's congruent to 6.0, while preincubation with 40 nM 3HE2 reveals specific binding peaks at EpH's of congruent to 6.0 and 7.5-8.0. Postmenopausal uterine cytosol preincubated with either 2 or 40 nM 3H-E2 on IEF reveals EpH = 5.8-6.0 binding only. Post-labeling of IEF fractions with 20 nM 3HE2 demonstrates one peak at EpH 5.8-6.0 in postmenopausal tissue and two peaks (5.8-6.2 and 7.5-8.0) in premenopausal tissue. Scatchard analysis of postmenopausal cytosol demonstrates a single population of binding sites with a dissociation constant (Kd) of 10(-10) M. Premenopausal cytosol on Scatchard analysis contains two estradiol binding populations with Kd's of 10(-10) and 10(-9) M. The data suggest that the 10(-10) M E2 binding population has a EpH of 5.8-6.2, while the 10(-9) M component has an EpH of 7.5-8.0.     

Characterization of the two eIF4A-binding sites on human eIF4G-1

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. eIF4G has two binding sites for the RNA helicase eIF4A, one in the central domain and one in the COOH-terminal domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fragments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 did not interfere with eIF4A binding to the central site. Interestingly, at the same concentration of free eIF4A, more eIF4A was bound to an eIF4G fragment containing both eIF4A sites than the sum of binding to fragments containing the single sites, indicating cooperative binding. Binding of eIF4A to an immobilized fragment of eIF4G containing the COOH-terminal site was competed by a soluble eIF4G fragment containing the central site, indicating that a single eIF4A molecule cannot bind simultaneously to both sites. The association rate constant, dissociation rate constant, and dissociation equilibrium constant for each site were determined by surface plasmon resonance and found to be, respectively, 1.2 x 10(5) m(-1) s(-1), 2.1 x 10(-3) s(-1), and 17 nm for the central site and 5.1 x 10(3) m(-1) s(-1), 1.7 x 10(-3) s(-1), and 330 nm for the COOH-terminal site.     

The interaction of secretin with pancreatic membranes.

1. 125I-labelled secretin bound rapidly and specifically to membranes from cat pancreas. Binding of labelled hormone was competitively inhibited by unlabelled secretin in the same range of concentrations that stimulated pancreatic adenylate cyclase in these membranes. The dissociation constant of the membrane binding sites for unlabelled secretin as evaluated by these displacement experiments was 4.1-10(-9) M and the number of binding sites 1.0 pmol per mg of membrane protein. 2. Studies using different concentrations of [125I]secretin (at a constant ratio of labelled to unlabelled hormone) revealed a similar value of 4-4-10(-9) M for the dissociation constant. 3. Both the association and dissociation rate constants of [125I]secretin binding were temperature sensitive; the dissociation rate constant increased more rapidly with increase in temperature. The ratio k-1/k+1 (at 22 degrees C) gave a dissociation constant of 3.7-10(-9)M which agrees closely with the figure obtained from equilibrium data. These data indicate that 125I-labelled secretin and unlabelled secretin bind to the same binding site on pancreatic membranes, with high affinity. 4. Unlabelled secretin stimulated pancreatic adenylate cyclase with an apparent Km of 8.4-10(-9) M, while [125I]secretin apparently did not stimulate the adenylate cyclase. Together with the binding data this might suggest that different portions of the secretin molecule are responsible for binding and adenylate cyclase activation. 5. Studies on the specificity of [125I]secretin binding carried out with various peptide hormones (glucagon, human gastrin, pancreozymin and caerulein) which are all inefficient in stimulating pancreatic fluid secretin, showed that these hormones have no influence on the binding of [125I]secretin. In contrast, vasoactive intestinal polypeptide, which stimulates pancreatic fluid and bicarbonate secretion, showed a competitive inhibition of secretin binding to the plasma membrane preparation.     

Interaction of vitamin K-dependent protein Z with thrombin. Consequences for the amidolytic activity of thrombin and the interaction of thrombin with phospholipid vesicles

Protein Z is a vitamin K-dependent protein of unknown function present in normal bovine plasma at a concentration of approximately 0.1 microM. Quantitative affinity chromatographic studies using diisopropylphosphoryl (DIP)-thrombin-Affi-Gel 10 as the affinity matrix and free DIP-thrombin as the competitor demonstrated that protein Z interacts with DIP-thrombin with a dissociation constant of 0.15 +/- 0.05 microM. Binding was independent of Ca2+. Protein C and factor IX, other vitamin K-dependent clotting proteins with the same domain structure as that of protein Z, did not interact with immobilized DIP-thrombin under these conditions; and factor X interacted with an affinity 20-fold lower than that for protein Z. The Michaelis constant, Km, for hydrolysis of pyro-Glu-Pro-Arg-p-nitroanilide by thrombin was increased 1.8-fold, from 130 to 230 microM, as a result of the binding of protein Z and the Km for H-Val-Leu-Arg-p-nitroanilide 1.4-fold, from 390 to 560 microM. From these kinetic studies, a dissociation constant of 0.11 +/- 0.04 microM was calculated for the binding of protein Z to alpha-thrombin. Protein Z bound to large phospholipid vesicles (25% phosphatidylserine, 75% phosphatidylcholine) with a dissociation constant of 0.39 +/- 0.16 microM at a phospholipid to protein ratio of 82 mol of phospholipid/mol of protein Z at saturation. In the presence of protein Z thrombin associated with phospholipid vesicles, whereas thrombin did not interact with phospholipid vesicles in the absence of protein Z. These studies, therefore, demonstrate a physiologically relevant interaction between protein Z and thrombin. They also suggest a mechanism whereby thrombin is localized to an injury site by virtue of its interaction with protein Z bound to phospholipid surfaces.     

Characterization of azadirachtin binding to Sf9 nuclei in vitro

[22,23-(3)H(2)]dihydroazadirachtin was incorporated by Sf9 cells in culture and was bound specifically to the nuclear fraction. The observed association constant of the binding of the radioligand to a purified nuclear fraction was determined to be 0.037 +/- 0.008 min(-1) using a one-phase exponential association equation, and binding appeared to be to a single population of sites. The binding was essentially irreversible, and the dissociation constant was estimated to be 0.00065 +/- 0.00013 min(-1). An association rate constant of 7.3 x 10(6) M(-1) min(-1) was calculated from these data. Binding was saturable, and the receptor number and affinity were determined as B(max) = 23.87 +/- 1.15 pmol/mg protein, K(d) = 18.1 +/- 2.1 nM. The order of potency of semisynthetic azadirachtin analogues for competition for the binding site was as follows (IC(50) in parentheses): azadirachtin (1.55 x 10(-8) M) > dihydroazadirachtin (3.16 x 10(-8) M) > dansyl dihydroazadirachtin (7.40 x 10(-8) M) > DNP-azadirachtin (7.50 x 10(-8) M) > biotin dihydroazadirachtin (1.27 x 10(-7) M) > 11-methoxy 22,23-dihydroazadirachtin (6.67 x 10(-7) M). [Originally published in Volume 34, Archives of Insect Biochemistry and Physiology, 34:461-473 (1997).] Copyright 1997 Wiley-Liss, Inc.     

Effect of lipid particle size on association of apolipoproteins with lipid

Triolein particles stabilized with egg yolk phosphatidylcholine monolayer were prepared with two different diameters: 26.7 +/- 3.9 and 229 +/- 80 nm. All the phosphatidylcholine molecules in those particles were readily digested by phospholipase A2 while only the molecules in the outer leaflet of phosphatidylcholine unilamellar vesicles were hydrolyzed under the same conditions. Binding of human plasma apolipoproteins A-I, A-II, C-II, and C-III2 to the particles was studied by two independent techniques: (i) rapid gel permeation chromatography and (ii) ultracentrifugation. All four apolipoproteins bound to the small and large particles in a saturable manner without altering their gross structure, and were displaced by equivalent molecules. The dissociation constant of apolipoprotein A-I for the large particle was 3.17 X 10(-6) M and 4.24 X 10(-6) M by methods (i) and (ii), respectively. These values were more than 10-fold greater than those for the small particles (2.0 X 10(-7) and 1.6 X 10(-7) M, respectively). In contrast, apolipoproteins A-II, C-II, and C-III2 bound to the large particles as strongly as to the small particles with dissociation constants of 2.4-6.8 X 10(-7), 4.5-10.7 X 10(-7), and 5.3-10.7 X 10(-7) M, respectively. The maximum binding level was of a similar order for each of the four apolipoproteins with both lipid particles when they were compared on the basis of amino acids per phospholipid. These results suggest that the apolipoproteins share common binding sites on the lipid particles, and are consistent with the characteristic distribution of apolipoproteins A-I and C among various classes of lipoproteins in plasma.     

Characterization of receptors for immune interferon in U937 cells with 32P-labeled human recombinant immune interferon

Recombinant human immune interferon (HuIFN-gamma) was labeled with [gamma-32P]ATP and cyclic-AMP-dependent protein kinase from bovine heart to a specific radioactivity of 11,000 Ci/mmol. At least two molecules of phosphate were incorporated per molecule of interferon. The binding of [32P]HuIFN-gamma to human U937 histiocytic lymphoma cells was time dependent, and displaceable by HuIFN-gamma but not by HuIFN-alpha A or HuIFN-beta. The specific binding was saturable with less than 10% nonspecific binding. The dissociation constant of [32P]HuIFN-gamma for U937 interferon receptors was calculated to be 1.5 X 10(-10) M with a total of 1,800 binding sites/cell. Dissociation of bound [32P]IFN-gamma at 24 degrees C exhibited two distinct rates. A fast dissociation with a specific rate constant of 0.141 min-1, and a slow dissociation with a specific rate constant of 0.0027 min-1. The Kd for [32P]HuIFN-gamma was calculated from kinetic constants to be 5.4 X 10(-10) M.     

Interaction of plasmin with endothelial cells.

Interaction of human plasmin with a monolayer culture of mini-pig aortic endothelial cells was studied by using the 125I-labelled enzyme. The binding of plasmin was time- and concentration-dependent. Equilibrium between bound and free enzyme was obtained within 90s, and Scatchard analysis indicated a high- and a low-affinity population of binding sites of approx. 1.24 X 10(4) sites/cell having a Kd of 1.4 X 10(-9) M and 7.2 X 10(4) sites/cell with a Kd of 2 X 10(-8) M respectively. Plasmin, bound to cell, was spontaneously released within 2 min, suggesting a rapid equilibrium. Chemical modification of the enzyme with phenylmethanesulphonyl fluoride or pyridoxal 5'-phosphate revealed that neither the active centre nor the heparin-binding site of plasmin was involved in the interaction with the endothelial cell. In terms of endothelial-cell receptors, the binding sites of cells for plasmin and thrombin were different: the two enzymes did not compete with each other, and the pretreatment of cells with neuraminidase or chondroitin ABC lyase resulted in a 50% decrease of thrombin or plasmin binding respectively. Arachidonic acid incorporated into phospholipids of the cell was released by plasmin, but a change in the rate of prostacyclin formation was not measurable. The interaction of plasmin with endothelial cells seems to be specific in the fibrinolytic system, since plasminogen did not bind to these cells under similar conditions.     

Solubilization of an alpha-bungarotoxin-binding component from rat brain.

Binding of [125I]-alpha-bungarotoxin to rat brain was investigated. Picomole quantities of specific toxin binding sites per gram of fresh tissue were found in particulate preparations as well as detergent extracts of whole brain. The toxin-binding macromolecules can be solubilized in low concentrations of Triton X-100. Specific binding occurs to a single class of sites with a dissociation constant of 5.6 X 10(-11) M. The association rate constant in 10 mM sodium phosphate, pH 7.4, was determined to be 6.8 X 10(5) M-1 s-1; the half-life of the complex was found to be 5.1 h, corresponding to a dissociation rate constant of 3.8 X 10(-5) s-1. The binding macromolecules resemble peripheral nicotinic acetylcholine receptors in toxin binding kinetics, solubility, isoelectric point, and hydrodynamic properties.     

An assay for beta-adrenergic receptors in isolated human fat cells

The beta-adrenergic receptors have been characterized in isolated human adipocytes using a potent beta-adrenergic antagonist (-)-[3H]dihydroalprenolol. Binding of (-)-[3H]dihydroalprenolol to isolated fat cells was stereospecific and saturable, the maximum number of binding sites calculated being 7.8 +/- 2.2 pmol of bound ligand/10(7) cells, corresponding to 450,000 binding sites/cell. The dissociation constant was estimated to be 2.7 +/- 1.1 nM. The results with competition-inhibition experiments using beta-adrenergic agonists and antagonists indicated that the binding sites in isolated adipocytes were predominantly of the beta1-subtype; about 80% of the receptors were of this type. With the present method, specific beta-adrenergic receptor number and affinity in isolated human adipocytes could be determined in about 1 g of human adipose tissue.