A microtechnique for quantification of detergent-solubilized muscarinic and nicotinic acetylcholine receptors using a semi-automated cell harvestor

A specific method for the rapid assay of muscarinic acetylcholine receptors (mAChR), either detergent-solubilized or in neuroblastoma cells, is described. This method is also applicable to the assay of nicotinic acetylcholine receptors. The procedure employs a cell harvestor and microtiter plates, and has the advantage of requiring small quantities of radioligand, microgram quantities of detergent-solubilized cholinergic receptor or less cells. The binding parameters such as the equilibrium dissociation constants (Kd) of mAChR and nicotinic acetylcholine receptor (nAChR) and inhibition constants (Ki) for antagonists determined by the present method are in excellent agreement with values determined by other methods. This assay procedure for mAChR and nAChR should facilitate the rapid screening of cholinergic agonists/antagonists and also the further purification and characterization of mAChR.     

Inhibition of enzymatic activity of alpha-thrombin by low molecular weight synthetic inhibitor]

The effect of the organophosphoric inhibitor, SA-152, on the fibrinogen-coagulating and TAME-esterase activity of bovine alpha-thrombin was studied. The irreversible inhibition constants (k11 = 1.1 x 10(4) M-1.min-1,Ki = 0.7 x 10(-4) M, k2 = 0.8 min-1 towards the coagulating activity and kII = 0.7 x 10(4) M-1.min-1, Ki = 0.3 x 10(-4) M, k2 = 0.2 min-1 towards the esterase activity) were determined. The SA-152 inactivated alpha-thrombin was dialyzed and incubated with 0.5 M and 2.5 M NaCl and 10 mM TAME. There was no reconstitution of activity of the SA-152 modified alpha-thrombin after dialysis and treatment with high concentrations of NaCl and TAME. Heparin interactions with the anion-binding site of the high molecular weight recognition center in the alpha-thrombin molecule did not significantly influence the values of the kinetic constants for the enzyme inhibition by SA-152. This finding is consistent with the hypothesis on the irreversible binding of SA-152 in the active center of the enzyme.     

In vitro characterization of skeletal muscle beta-adrenergic receptors coupled to adenylate cyclase.

[3H]Dihydroalprenolol, a potent beta-adrenergic antagonist, was used to identify the adenylate cyclase-coupled beta-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed by [3H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [3H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [3H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions. The binding of [3H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K1) and dissociation (K2) rate constants of 2.21 . 10(7) M-1 . min-1 and 3.21 . 10(-1) min-1, respectively, were obtained. The dissociation constant (Kd) of 15 mM for [3H]dihydroalprenolol obtained from saturation binding data closely agreed with the Kd derived from the ratio of dissociation and association rate constants (K2/K1). Several beta-adrenergic agents known to be active on intact skeletal muscle also competed for [3H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [3H]dihydroalprenolol binding sites with a potency of isoproterenol greater than epinephrine greater than norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (-)-isomers being more potent than (+)-isomers. Phenylephrine, an alpha-adrenergic agonist, showed no effect either on [3H]dihydroalprenolol binding or on adenylate cyclase. Known beta-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [3H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The Ki values for the antagonists determined from inhibition of [3H]dihydroalprenolol binding agreed closely with the Ki values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [3H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the beta-adrenergic receptor.     

A high affinity, highly selective ligand for the delta opioid receptor: [3H]-[D-Pen2, pCl-Phe4, d-Pen5]enkephalin

Binding characteristics of a new, conformationally constrained, halogenated enkephalin analogue, [3H]-[D-penicillamine2, pCl-Phe4, D-penicillamine5]enkephalin ([3H]pCl-DPDPE), were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C determined a dissociation constant (Kd) of 328 +/- 27.pM and a receptor density (Bmax) of 87.2 +/- 4.2 fmol/mg protein. Kinetic studies demonstrated biphasic association for [3H]pCl-DPDPE, with association rate constants of 5.05 x 10(8) +/- 2.5 x 10(8) and 0.147 +/- 10(8) +/- 0.014 x 10(8) M-1 min-1. Dissociation was monophasic with a dissociation rate constant of 2.96 x 10(-3) +/- 0.25 x 10(-3) min-1. The average Kd values determined by these kinetic studies were 8.4 +/- 2.7 pM and 201 +/- 4 pM. Competitive inhibition studies demonstrated that [3H]pCl-DPDPE has excellent selectively for the delta opioid receptor. [3H]pCl-DPDPE binding was inhibited by low concentrations of ligands selective for delta opioid receptor relative to the concentrations required by ligands selective for mu and kappa sites. These data show that [3H]pCl-DPDPE is a highly selective, high affinity ligand which should be useful in characterizing the delta opioid receptor.     

Solubilisation and Characterisation of a Putative Quisqualate-Type Glutamate Receptor from Chick Brain

The brains of 1-day-old chicks were shown to be a rich source of binding sites with the pharmacological characteristics expected of a quisqualate-type glutamate receptor. alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) bound with KD and Bmax values, measured at 0 degree C in the presence of the chaotrope potassium thiocyanate, of 55 nM and 2.6 pmol/mg protein. The regional localisations of [3H]AMPA and [3H]kainate binding sites were manifestly different. The membrane-bound [3H]AMPA binding sites were efficiently solubilised by N-octyl-beta-D-glucopyranoside (1%) in the presence of 0.2 M thiocyanate. In the detergent extract the affinity was 69 nM and there was an apparent increase in the number of sites (Bmax, 4.6 pmol/mg protein). The rank order of potency for competitive ligands in displacing [3H]AMPA binding was quisqualate approximately AMPA greater than 6-cyano-7-nitroquinoxaline-2,3-dione greater than L-glutamate greater than kainate and was identical for the membrane-bound and solubilised sites. Dissociation was biphasic with rate constants of 0.117 min-1 and 0.015 min-1. The association rate constants for [3H]AMPA at the solubilised sites were 1.45 x 10(6) M-1 min-1 and 6.55 x 10(6) M-1 min-1. The kinetically derived KD values were 80.7 nM and 2.3 nM. The detection of higher affinity binding sites by kinetic analysis but not by equilibrium binding may be explained by the greater sensitivity of dissociation data to small populations of high-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The kinetics of hemostatic enzyme-antithrombin interactions in the presence of low molecular weight heparin

The kinetics of inhibition of four hemostatic system enzymes by antithrombin were examined as a function of heparin concentration. Plots of the initial velocity of factor Xa-antithrombin or plasmin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb and subsequent plateau regions. In each case, the kinetic profile is closely correlated with the concentration of the heparin . antithrombin complex formed within the reaction mixture. A decrease in the velocity of inhibition is not observed at high levels of added mucopolysaccharide despite the generation of significant quantities of heparin-enzyme interaction products. The second-order rate constants for the neutralization of factor Xa or plasmin by the mucopolysaccharide . inhibitor complex are 2.4 x 10(8) M-1 min-1 and 4.0 x 10(6) M-1 min-1, respectively. These parameters must be contrasted with the similarly designated constants obtained in the absence of heparin which are 1.88 x 10(5) M-1 min-1 and 4.0 x 10(4) M-1 min-1, respectively. Plots of the initial velocity of the factor IXa-antithrombin or the thrombin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb, pseudoplateau, descending limb, and final plateau regions. In each case, the ascending limb and pseudoplateau are closely correlated with the concentration of heparin c antithrombin complex formed within the reaction mixture. Furthermore, the descending limb and final plateau of these two processes coincide with the generation of increasing amounts of the respective mucopolysaccharide-enzyme interaction products. The second-order rate constants for the neutralization of factor IXa or thrombin by the heparin . antithrombin complex are 3.0 x 10(8) M-1 min-1 and 1.7 x 10(9) M-1 min-1, respectively. The second-order rate constants for the inhibition of mucopolysaccharide-factor IXa or mucopolysaccharide-thrombin interaction products by the heparin . antithrombin complex are 2.0 x 10(7) M-1 min-1 and 3.0 x 10(8) M-1 min-1, respectively. These kinetic parameters must be contrasted with similarly designated constants obtained in the absence of mucopolysaccharide which are 2.94 x 10(4) M-1 min-1 and 4.25 x 10(5) M-1 min-1, respectively. Thus, our data demonstrate that binding of heparin to antithrombin is required for the mucopolysaccharide-dependent enhancement in the rates of neutralization of thrombin, factor IXa, factor Xa, or plasmin by the protease inhibitor. Furthermore, a careful comparison of the various constants suggests that the direct interaction between heparin and antithrombin may be largely responsible for the kinetic effect of this mucopolysaccharide.     

Muscarinic Cholinergic Receptors in Human Foetal Brain: Characterization and Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Frontal Cortex

Twenty-two frontal cortices from normal human foetal brains of gestational ages ranging from 16 to 40 weeks and five postnatal brains ranging from 5 to 50 years were analysed for the ontogeny of muscarinic receptors using [3H]quinuclidinyl benzilate (QNB) as the ligand. QNB binding sites were shown to be stable up to 4 1/2 months of storage at -70 degrees C. QNB binding was characterized in frontal cortices of 28-week-old foetal brains as muscarinic receptors by the following criteria: (1) it was localised mainly in particulate fraction; (2) binding was saturable at a concentration of 1.5 nM; (3) the cholinergic antagonists atropine and scopolamine competed for the binding, with IC50 values of 1 and 0.8 nM, respectively. The agonists oxotremorine, carbachol, and pilocarpine gave IC50 values of 1, 15 and 18 microM, respectively. Nicotinic receptor ligands and noncholinergic drugs could not compete for the binding. Bimolecular association and dissociation rate constants for the reversible binding are 6.23 X 10(8) M-1 X min-1 and 2.0 X 10(-2) X min-1, respectively. The equilibrium dissociation constant is 33 pM. The KD obtained by saturation binding data is 103 pM. Ontogeny of muscarinic receptors showed three distinct phases: In phase I, they appear between 16 and 18 weeks [average concentration 109 fmol/mg protein of total particulate fraction (TPF)] and slowly increase up to 20 weeks (average concentration 147 fmol/mg protein TPF). Phase II is a lag period between 20 and 24 weeks at which time receptor concentration does not change perceptibly (average concentration (67 fmol/mg protein TPF).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the 1 alpha,25-dihydroxyvitamin D3 receptor from rat intestinal cytosol.

The 1 alpha,25-dihydroxyvitamin D3 receptor from rat intestinal cytosol has been partially characterized. Sucrose density gradient sedimentation and analytical gel filtration analyses of this receptor yielded values of 3.1 S, 80,000, and 36 A for the sedimentation coefficient, molecular weight (Mr), and Stokes molecular radius (Rs), respectively. The receptor was found to be a protein by its susceptibility to protease but not nuclease digestion, and studies with N-ethylmaleimide and iodoacetamide revealed the presence of a reduced cysteine residue near the ligand binding site of the receptor. Kinetic and equilibrium binding studies showed an equilibrium dissociation constant of 7.4 x 10(-10) M (4 degrees C), an association rate constant of 1.7 x 10(7) M-1 min-1 (0 degrees C) and a dissociation rate constant of 7.2 x 10(-4) min-1 (4 degrees C, t1/2 = 16 h).     

Interaction of di-iodinated 125I-labelled alpha-bungarotoxin and reversible cholinergic ligands with intact synaptic acetylcholine receptors on isolated skeletal-muscle fibres from the rat.

1. Intact synaptic acetylcholine receptors on freshly isolated rat skeletal-muscle fibres were characterized by their interaction with di-iodinated 125I-labelled alpha-bungarotoxin, acetylcholine and other cholinergic ligands at room temperature (22 deggrees C). 2. The time course and concentration dependence of 125I-labelled alpha-bungarotoxin association conformed to a bimolecular mechanism. In time-course experiments with different concentrations of 125I-labelled alpha-bungarotoxin (1.4--200 nM) the bimolecular-association rate constant, k + 1, was (2.27 +/- 0.49) x 10(4)M-1.S-1 (mean +/- S.D., N = 10). In concentration-dependence experiments, k + 1 was 2.10 x 10(4)M-1.S-1 and 1.74 x 10(4) M-1.S-1 with 10 and 135 min incubations respectively. In association experiments the first-order rate constant was proportional to the 125I-labelled alpha-bungarotoxin concentration. 125I-Labelled alpha-bungarotoxin dissociation was first order with a dissociation constant, k-1, less than or equal to 3 x 10(-6)S(-1) (half-life greater than or equal to 60 h.) The results indicated a single class of high-affinity toxin-binding sites at the end-plate with an equilibrium dissociation constant, Kd, equal to or less than 100 pM. The number of toxin-binding sites was (3.62 +/- 0.46) x 10(7) (mean +/- S.D., n = 22) per rat end-plate. 3. The apparent inhibitor dissociation constants, Ki, for reversible cholinergic ligands were determined by studying their effect at equilibrium on the rate of 125I-labelled alpha-bungarotoxin binding. There was heterogeneity of binding sites for cholinergic ligands, which were independent and non-interacting with antagonists. In contrast agonist affinity decreased with increasing receptor occupancy. Cholinergic ligands in excess inhibited over 90% of 125I-labelled alpha-bungarotoxin binding. 4. Cholinergic ligand binding was accompanied by an increase in entropy, which was greater for the agonist carbachol (delta So = +0.46 kJ.mol-1.K-1) than the antagonist tubocurarine (delta So = +0.26 kJ.mol-1.K-1). 5. The entropy and affinity changes that accompanied agonist binding suggested that agonists induced significant conformational changes in intact acetylcholine receptors. 6. The affinity and specificity of 125I-labelled alpha-bungarotoxin and tubocurarine binding to synaptic acetylcholine receptors from slow and fast muscle fibres were the same. 7. The study of binding only requires milligram amounts of tissue and may have application to other neurobiological studies and to the study of human neuromuscular disorders.     

Kinetics of basic fibroblast growth factor binding to its receptor and heparan sulfate proteoglycan: a mechanism for cooperactivity.

Basic fibroblast growth factor (bFGF) binds to cell surface receptor (CSR) proteins and to heparan sulfate proteoglycans (HSPG). On the basis of equilibrium dissociation constants (Kd), the CSR has been considered a "high-affinity" binding site and HSPG a "low-affinity" site. We measured the apparent individual on and off rate constants (kon and koff) for bFGF binding to these two sites on intact cells and to each class of binding site in the absence of the other. While the kon's for CSR and HSPG on intact cells were not statistically different (konC = 2.27 x 10(8) M-1 min-1; konH = 0.90 x 10(8) M-1 min-1), the koff for the HSPG was 22.7-fold greater than that for the CSR (koffC = 0.003 min-1; koffH = 0.68 min-1). Thus, the difference in Kd's appears to result from the faster rate at which bFGF is released from the HSPG sites compared to the CSR. The kon's for isolated CSR and HSPG, and the koff for isolated HSPG, did not differ significantly from those for intact cells konC = 2.50 x 10(8) M-1 min-1; konH = 0.92 x 10(8) M-1 min-1; koffH = 0.095 min-1). However, the off rate for isolated CSR (koffC = 0.048 min-1) was statistically indistinguishable from the off rate for HSPG and 16-fold greater than the off rate for CSR on intact cells. The "high-affinity" binding of bFGF to intact cells probably refers only to a complex of bFGF with both CSR and HSPG, and not to the CSR alone.     

Kinetics of inhibition of calf intestinal adenosine deaminase by (+)- and (-)-erythro-9-(2-hydroxy-3-nonyl)adenine.

The enantiomers of erythro-9-(2-hydroxy-3-nonyl)adenine [(+)- and (-)-EHNA) bound to adenosine deaminase (ADA) at pH 7 with concomitant changes in the optical properties of the enzyme. The association rate constant for (+)-EHNA was 2.9 x 10(6) M-1 s-1 and that for (-)-EHNA was 6.4 x 10(6) M-1 s-1. The dissociation of (-)-EHNA.ADA or (+)-EHNA.ADA in the presence of excess coformycin was monitored by the quenching of enzyme fluorescence as coformycin.ADA was formed. The dissociation rate constants of (+)- and (-)-EHNA.ADA were 0.0054 s-1 and 2.7 s-1, respectively. A similar value for the dissociation rate constant (0.005 s-1) for (+)-EHNA.ADA was calculated from the time course for the appearance of catalytic activity after dilution of (+)-EHNA.ADA into 100 microM adenosine. The Ki values of ADA for (+)- and (-)-EHNA were similar to the dissociation constants calculated from the ratio of the respective dissociation and association rate constants. The biphasic time-dependent inhibition of the catalytic activity of ADA by (+/- )-EHNA [Frieden, C., Kurz, L. C., & Gilbert, H. R. (1980) Biochemistry 19, 5303-5309] was confirmed. However, the catalytic activity of ADA was inhibited monophasically by (+)-EHNA. Thus, the biphasic nature of the time course for inhibition of ADA by (+/- )-EHNA was the result of the presence of both enantiomers of the inhibitor in this assay. These kinetic data were interpreted in terms of single-step mechanisms for binding of (+)- and (-)-EHNA.     

Identification and characterization of the beta-adrenergic receptor on neuroblastoma × glioma hybrid NG108-15 cells

1. Using [3H]DHA and unlabeled L-alprenolol, a substantial amount of over 64% specific binding of beta-adrenergic receptor has been identified on the neuroblastoma x glioma hybrid NG108-15 cell, which has been proven to display numerous functional characteristics of intact neurons. 2. Beta-adrenergic receptor binding on intact NG108-15 cells does not change significantly upon morphological differentiation, induced by 1 mM dibutyryl cyclic AMP (dBcAMP). 3. The [3H]DHA binding on intact NG108-15 cells is rapid, saturable, and reversible, having a t1/2 of 1.0 min for association and 3.5 min for dissociation. 4. The affinity constant (Kd) and maximum binding capacity (Bmax) for binding of [3H]DHA to beta-adrenergic receptors on NG108-15 cells have been estimated by Scatchard plot analysis to be 2.5 and 0.23 nM, respectively. Further analysis indicates a single class of receptors for [3HDHA binding on NG108-15 cells. 5. Studies on kinetic properties have revealed on-rate (K + 1) and off-rate (K - 1) constants of 0.7 X 10(-9) M min-1 and 0.19 min-1, respectively. Further, the IC50 value and inhibition constant (Ki) for unlabeled L-alprenolol to inhibit [3HDHA binding on NG108-15 cells have been estimated to be 10(-5) and 8.9 X 10(-6) M, respectively. 6. The rank-order potency of catecholamine agonists, (-)ISO greater than (+)ISO greater than EPI greater than NE, reveals the presence of type 2 receptor for the beta-adrenergic binding on both differentiated and undifferentiated NG108-15 cells. 7. The present study indicates that the clonal neuroblastoma x glioma hybrid NG108-15 cell line possesses substantial amounts of beta-adrenergic receptors with characteristics similar to those on neuronal cells.     

The Intact Human Neuroblastoma Cell (SH-SY5Y) Exhibits High-Affinity [3H]Pirenzepine Binding Associated with Hydrolysis of Phosphatidylinositols

The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 +/- 1.4 X 10(6) M-1 min-1 and 1.1 +/- 0.06 X 10(-1) min-1, respectively. Saturation experiments revealed a single class of binding sites (nH = 1.1) for the radioligand with a total binding capacity of 160 +/- 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency was atropine sulfate greater than pirenzepine greater than AF-DX 116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](-)-quinuclidinyl benzilate and its quaternary derivative [3H](-)-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.     

Characterization of binding of a specific antagonist, [3H]-SQ 29,548, to soluble thromboxane A2/prostaglandin H2 (TP) receptors in human platelet membranes.

Binding of [3H]-SQ 29,548 was characterized to soluble thromboxane A2/prostaglandin H2 (TP) receptors from human platelet membranes as a means of examining ligand-receptor interactions outside the lipophilic environment of the cell membrane. Kinetic determination revealed a rate of ligand-receptor association of 1.4 x 10(7) +/- 0.2 M-1 x min-1 and a rate of dissociation of 0.5 +/- 0.07 min-1. The resultant equilibrium affinity constant was 36.3 +/- 5.8 nM. Saturation binding analysis revealed a single class of [3H]-SQ 29,548 binding sites with an affinity constant of 39.7 +/- 4.3 nM and a B(max) of 1735.7 +/- 69.1 fmol/mg protein. Specific [3H]-SQ 29,548 binding was inhibited by specific TP receptor antagonists and agonists in a rank order of potency similar to that seen in platelet membranes: SQ 33,961 much greater than SQ 29,548 greater than BM 13,505 greater than or equal to U 46619 greater than BM 13,177. PGD2, PGE2 and PGI2 did not appreciably inhibit the specific binding of [3H]-SQ 29,548. These data indicate that [3H]-SQ 29,548 binding to soluble human platelet TP receptors was specific, saturable, and reversible.     

Characterization of intestinal smooth muscle responses and binding sites for endothelin.

The contractile activity of and binding sites for endothelin-1 (ET-1) were investigated in isolated guinea-pig ileal longitudinal smooth muscle (GPILM). ET-1 produced concentration-dependent contractions of GPILM that either slowly subsided in the continued presence of ET-1 or rapidly subsided following washing of the tissue. The ED50 value for ET-1 contractions was 4.2 +/- 1.3 x 10(-9) M. The removal of extracellular calcium or pretreatment with nifedipine produced a complete inhibition of the contractions to ET-1. The IC50 value of nifedipine for inhibition of ET-1 mediated contractions was 3.0 +/- 0.8 x 10(-8) M. ET-1 produced a marked prolonged homologous desensitization of its contractile response but did not affect the responses mediated by carbachol, histamine, serotonin, substance P, and PLA2. High-affinity binding sites for 125I-labelled ET-1 were identified on microsomal membranes prepared from GPILM with Kd and Bmax values obtained by Scatchard analysis of 3.5 +/- 0.6 x 10(-10) M and 2138 +/- 159 fmol/mg protein, respectively. The binding of 125I-labelled ET-1 to GPILM microsomes was characterized by a rapid association (kob value of 0.077 min-1 at a radioligand concentration of 0.45 nM and an extremely slow dissociation (k1 value of 0.011 min-1; t1/2 value of 793 min). The binding was unaffected by the calcium channel antagonists nifedipine, verapamil, and diltiazem (10(-6) M); the receptor antagonists phenoxybenzamine, atropine, and naloxone (10(-6) M) and propranolol; and the peripheral benzodiazepine receptor antagonists Ro 5-4864 and PK 11195 and psychotomimetic drug phencyclidine (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of very low density lipoprotein with chicken oocyte membranes

The interaction of hen 125I-VLDL (very low density lipoprotein) with chicken oocyte membranes was characterized using a rapid sedimentation assay. Equilibrium and kinetic studies showed an apparent dissociation constant (Kd) 8.7-9.1 x 10(-8) M or 43.5-45.5 micrograms VLDL protein/ml. Binding capacity was 2.0 micrograms VLDL protein/mg membrane homogenate protein. The apparent rate constants were k1 = 2.4 x 10(5) M-1 min-1 and k2 = 2.1 x 10(-2) min-1. Specific binding required the presence of divalent cations. Whereas binding was completely restored after treatment with EDTA by the addition of MN++, only 60% of binding was restored using Ca++.     

Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin.

We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable enterotoxin receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously expresses STa binding sites. Scatchard analysis of displacement binding studies revealed a single STa binding site with an affinity (Ki) of 97 pM in 293-STaR compared with 55 pM in T84 cells. Saturation isotherms of STa binding gave a Kd of 94 pM for the cloned receptor expressed in 293 cells and 166 pM for the receptor present in T84 cells. Kinetic measurements of STa binding to 293-STaR gave an association rate constant, K1, of 2.4 x 10(8) M-1 min-1 and a dissociation rate constant, K2, of 0.016 min-1. The half-time of dissociation was 43 min, and the Kd calculated from the ratio of the kinetic constants was 67 pM. The pH profile of STa binding showed that the number of STa binding sites is increased 3-fold at pH 4.0 compared with pH 7.0, with no effect on binding affinity. A polyclonal antibody directed against the extracellular domain of STaR immunoprecipitated two proteins of approximately 140 and 160 kDa from both 293-STaR and T84 cells. Cross-linking of 125I-STa to 293-STaR cells resulted in the labeling of proteins with a molecular mass of approximately 153, 133, 81, 68, 56, and 49 kDa, the two smallest being the more abundant. Similar results have been reported for the STaR present on rat brush border membranes. These data suggest that the STaR-guanylyl cyclase identified by molecular cloning is the only receptor for STa present in T84 cells.     

Scintillation proximity assay as a high-throughput method to identify slowly dissociating nonpeptide ligand binding to the GnRH receptor

Many nonpeptide antagonists of the gonadotropin-releasing hormone (GnRH) receptor, as well as other drug targets, possess a broad range of dissociation kinetic rate constants. Current methods to accurately define kinetic rate parameters such as K(on) and K(off) are time and labor intensive, prompting the development of a screening assay to identify slowly dissociating compounds for follow-up rate constant determination. The authors measured inhibition binding constants (K(i)) for GnRH receptor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased K(i) values over time (Ki(30 min)/Ki(10 h) > 6). They used scintillation proximity assay technology to perform these binding experiments because this homogeneous assay does not have a fixed termination end point as does filtration binding, permitting successive readings to be taken from the same assay plate over an extended period of time. They also used a quantitative method of kinetic rate analysis to confirm that a large disparity between a compound's K(i) value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the K(i) ratio can be used to screen for and select compounds to test using more quantitative, albeit lower throughput methods to accurately define kinetic rate constants.     

A variant of human transferrin with abnormal properties.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.     

Kinetics of the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis.

The rate constants for the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis were determined by using a pre-steady-state kinetic approach. kon and koff for complex-formation and dissociation were 1 X 10(6)M-1 X S-1 and 8 X 10(-4)S-1 respectively. Ki was calculated as the ratio koff/kon = 8 X 10(-10)M, the binding of eglin to elastase was reversible and the inhibition mechanism was of the fully competitive type. The mechanistic properties of the system and the biological significance of the rate constants are discussed.