Characterization of [3H]Ro 5-4864 Binding Sites in Rat Vas Deferens

The presence of benzodiazepine binding sites in rat vas deferens was detected using [3H]Ro 5-4864 as a radioligand. The binding of [3H]Ro 5-4864 to the mitochondrial sites is saturable, reversible, and temperature and time dependent. The association rate constant (k1) was 8.7 +/- 0.7 x 10(7) M-1 min-1, and the dissociation rate constant (k-1) was 0.031 +/- 0.003 min-1. The dissociation constant (KD) determined by saturation binding was 5.22 +/- 0.56 nM. The density of binding was 4,926 +/- 565 fmol/mg of protein. The Hill coefficient of binding was 0.99 +/- 0.01, an indication that [3H]Ro 5-4864 binds to a single site. The [3H]Ro 5-4864 binding was inhibited competitively by Ro 5-4864 and 2-hydroxy-5-nitrobenzyl-6-thioguanosine and noncompetitively by PK 11195, nitrendipine, alpha,beta-methylene-ATP, and carboxyatractyloside and was not affected by clonazepam, dicyclohexylcarbodiimide, or protoporphyrin IX. Our data indicate that [3H]Ro 5-4864 binding sites are not identical to those labeled by PK 11195. These binding sites are modulated by the ADP/ATP mitochondrial carrier, and an interaction of dihydropyridines and [3H]Ro 5-4864 binding sites in rat vas deferens is suggested.     

Binding of [14,15-3H]14,15-dihydroforskolin to rat liver membranes: comparison with the stimulatory effect of forskolin on adenylate cyclase

The binding of [14,15-3H]14,15-dihydroforskolin ([3H]DHF) to rat liver membranes has been further characterized and was compared with the stimulatory effect of forskolin on adenylate cyclase. The binding equilibrium dissociation constant (KD) for 14,15-dihydroforskolin obtained in inhibition experiments was 0.6 microM, with a maximal binding capacity (Bmax) of 114 pmol/mg protein. A similar KD value (0.5 microM) was derived from kinetics studies that revealed very rapid association and dissociation reactions. For structure-activity relationship studies several forskolin derivatives were synthesized and tested for their ability to inhibit [3H]DHF binding and increase adenylate cyclase activity. Among the tested compounds, forskolin itself was the most potent agonist (K1 = 0.2 microM). Further modification of the molecule in position 7 and (or) 1 decreased or abolished its agonist properties in both adenylate cyclase and binding studies. [3H]DHF binding was not affected by several nucleotides, carbohydrates, lectins, and hormone receptor agonists including isoproterenol, glucagon, and adenosine, but the steroids 17-beta-estradiol, progesterone, and testosterone showed slight inhibitory effects at unphysiologically high concentrations. [3H]DHF binding and forskolin-stimulated adenylate cyclase were sensitive to heat and N-ethylmaleimide treatment. Forskolin protected adenylate cyclase against inactivation by heat but not by N-ethylmaleimide. Preincubation of the membrane with trypsin decreased [3H]DHF binding. The results presented in this study demonstrate that the binding sites identified with [3H]DHF have a high specificity for forskolin and provide evidence that these binding sites are involved in the stimulation of adenylate cyclase by forskolin.     

Identification of beta adrenergic receptors in rat hypothalamus.

(-)-[3H]-Dihydroalprenolol((-)[3H]DHA) binding in the rat hypothalamus appears to possess all the characteristics expected of physiologically relevant beta-adrenergic receptors. Binding of (-)-[3H]DHA to the hypothalamic sites was rapid (k1 = 1.3 X 10(-7) min-1) and also rapidly reversible. Binding was saturable at low concentrations of ligand (approximately 50-100 nM). The dissociation constant (KD) of (-)-[3H]DHA binding determined by equilibrium analysis was 19 nM. Binding displayed beta-adrenergic specificity. beta-Adrenergic agonists inhibited binding in the following order of potency: (-)-isoproterenol congruent to (-)-epinephrine greater than (-)-norepinephrine. Specific beta-adrenergic antagonists (-)-propranol and (-)-alprenolol inhibited binding at low concentrations (KD = 25-50nM) whereas the alpha-antagonist phentolamine inhibited binding at very high concentration (KD = 42 micron). Interactions of both agonists and antagonists with the sites showed stereoselectivity. The (-)-isomers of all beta-adrenergic agents tested were more potent than their respective (+)-isomers. These results suggest that specific receptor sites for beta-adrenergic catecholamines are present in rat hypothalamus.     

Evidence for a single forskolin-binding site in rat adipocyte membrane. Studies of [14,15-3H]dihydroforskolin binding and adenylate cyclase activation

[14,15-3H] Dihydroforskolin has been used as a tracer in the study of forskolin binding to adipocyte plasma membrane and the subsequent activation of adenylate cyclase (EC 4.6.1.1) of this membrane. The specific binding of radioactivity to the membrane was rapid, temperature-dependent, saturable, and readily reversible. The equilibrium dissociation reaction constant (KD) for the binding was 13 microM, with a maximum binding (Bmax) of 61 pmol of forskolin per mg of membrane protein. The Hill coefficient was 1.0. The bound [14,15-3H] dihydroforskolin was displaced by forskolin with rate constants of 0.07 X 10(6) M-1 min-1 and 1.2 min-1 for the association and dissociation reactions, respectively (30 degrees C). The equilibrium dissociation constant (KD) was approximately the same as the concentration that produced half-maximum activation (EC50) of the adenylate cyclase of rat adipocyte plasma membrane. There was a linear correlation between forskolin binding and adenylate cyclase activation. The results are consistent with the concept of a single class of binding site which binds forskolin. [14,15-3H] Dihydroforskolin appears to be a potentially useful tracer in the study of the mechanism of activation of the catalytic unit of adipocyte adenylate cyclase.     

Evidence for muscarinic cholinergic receptors in dog portal vein: binding of [3H]quinuclidinyl benzilate

Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [3H]quinuclidinyl benzilate (QNB) as a ligand. Specific [3H]QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (KD = 15.5 +/- 2.8 pM) with a Bmax of 110 +/- 14.7 fmol/mg protein. Scatchard and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 X 10(9) M-1 min-1 and 0.016 min-1, respectively, were calculated. The dissociation constant calculated from the equation KD = K-1/K+1 was 8.3 pM, such being in good agreement with the Scatchard estimate of KD (15.5 pM). Specific binding of [3H]QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, alpha-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [3H]QNB binding at 10 microM. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.     

Characterization of picomolar affinity binding sites for [125I]-human calcitonin gene-related peptide in rat brain and heart

We have characterized picomolar affinity binding sites for human calcitonin gene-related peptide (CGRP) in rat brain and heart (atria and ventricle) membranes. By saturation analysis, apparent dissociation constant (KD) values of high affinity sites for [125I]-human CGRP are 9 approximately 15 pM (brain), 34 pM (ventricle) and 85 pM (atria). Low affinity sites with KD values of about 50 nM are found in rat brain and ventricle, but not in atria. Human and rat CGRP potently inhibited [125I]-human CGRP binding to these high affinity sites with apparent inhibition constant (Ki) values comparable to their KD values. Salmon calcitonin marginally inhibited these binding with Ki values between 0.1 microM and 1 microM. Extremely potent cardiovascular and gastrointestinal actions of CGRP might be mediated through CGRP binding sites with picomolar affinity which are similar to those we characterized in this study.     

A Trifluoromethylphenyl Piperazine Derivative with High Affinity for 5-Hydroxytryptamine-1A Sites in Rat Brain

The inhibition of [3H]5-hydroxytryptamine [( 3H]5-HT) binding in rat brain by 1-[2-(3-bromoacetamidophenyl)ethyl]-4-(3-trifluoromethylphenyl) piperazine (BrAcTFMPP) and that by spiperone were compared. Spiperone inhibition of [3H]5-HT binding in cortex was consistent with displacement from two sites with dissociation constants (KD) of 24 nM (5-HT-1A site) and 19 microM (5-HT-1B site) for spiperone. BrAcTFMPP also discriminated two subpopulations of [3H]5-HT binding sites with dissociation constants of 0.5 nM and 146 nM for the compound. The proportion of high-affinity sites for each compound represented about 35% of the specific [3H]5-HT binding. In the presence of 1 microM spiperone, a concentration that saturates the 5-HT-1A sites while having a minimal effect on 5-HT-1B sites, BrAcTFMPP displaced [3H]5-HT from a single site with a KD for BrAcTFMPP of 145 nM. The inhibition of [3H]5-HT binding by spiperone in the presence of 30 nM BrAcTFMPP was best fit by a single-site model with a KD of 21 microM for spiperone. In corpus striatum, 5-HT-1A sites, as defined with spiperone, represented 15% of the specific [3H]5-HT binding and 30 nM BrAcTFMPP also blocked about 15% of the binding. A significant difference between spiperone and BrAcTFMPP was their affinity for 5-HT-2 receptors. BrAcTFMPP (KD = 41 nM) had an 80-fold lower affinity for these sites than spiperone (KD = 0.5 nM). Thus, BrAcTFMPP and spiperone discriminate the same two subpopulations of [3H]5-HT binding sites and BrAcTFMPP displays a high affinity and a selectivity for 5-HT-1A sites versus both 5-HT-1B and 5-HT-2 sites.     

Human aldehyde dehydrogenase: coenzyme binding studies

The binding of NADH and NAD+ to the human liver cytoplasmic, E1, and mitochondrial, E2, isozymes at pH 7.0 and 25 degrees C was studied by the NADH fluorescence enhancement technique, the sedimentation technique, and steady-state kinetics. The binding of radiolabeled [14C]NADH and [14C]NAD+ to the E1 isozyme when measured by the sedimentation technique yielded linear Scatchard plots with a dissociation constant of 17.6 microM for NADH and 21.4 microM for NAD+ and a stoichiometry of ca. two coenzyme molecules bound per enzyme tetramer. The dissociation constant, 19.2 microM, for NADH as competitive inhibitor was found from steady-state kinetics. With the mitochondrial E2 isozyme, the NADH fluorescence enhancement technique showed only one, high-affinity binding site (KD = 0.5 microM). When the sedimentation technique and radiolabeled coenzymes were used, the binding studies showed nonlinear Scatchard plots. A minimum of two binding sites with lower affinity was indicated for NADH (KD = 3-6 microM and KD = 25-30 microM) and also for NAD+ (KD = 5-7 microM and KD = 15-30 microM). A fourth binding site with the lowest affinity (KD = 184 microM for NADH and KD = 102 microM for NAD+) was observed from the steady-state kinetics. The dissociation constant for NAD+, determined by the competition with NADH via fluorescence titration, was found to be 116 microM. The number of binding sites found by the fluorescence titration (n = 1 for NADH) differs from that found by the sedimentation technique (n = 1.8-2.2 for NADH and n = 1.2-1.6 for NAD+).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Receptors for Substance P in Human Astrocytoma Cells: Radioligand Binding and Inositol Phosphate Formation

UC11 cells, derived from a human astrocytoma, have a high density of functional substance P receptors. Radioligand binding studies were conducted with the highly selective neurokinin-1 receptor ligand [3H][Sar9,Met(O2)11]-substance P. Kinetic binding experiments conducted at 4 degrees C yielded an association rate constant k1 of 1.86 x 10(7) M-1 min-1, a dissociation rate constant k-1 of 0.00478 min-1, and a calculated kinetic KD of 257 pM. Saturation binding experiments yielded average values of KD = 447 +/- 103 pM, Bmax = 862 +/- 93 fmol/mg of protein. This Bmax corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar9,Met(O2)11]-substance P yielded average values of KD = 491 +/- 48 pM and Bmax = 912 +/- 67 fmol/mg of protein. In [3H]inositol-labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20-fold within approximately 15 s of addition of substance P. This inositol trisphosphate formation was transient and had returned to baseline within the first 60-120 s. Inositol monophosphate formation, however, was linear for at least 2 h. Structure activity data on binding and inositol monophosphate formation confirmed the presence of a neurokinin-1 receptor subtype in these cells. Thus, the UC11 cell should be a useful model cell for delineating the physiological role of substance P receptors in astrocytes.     

Benzodiazepine receptors along the nephron: [3H]PK 11195 binding in rat tubules

Binding of [3H]PK 11195, an isoquinoline carboxamide derivative, was measured in microdissected tubule segments of rat nephron. High specific binding capacities (1.1-1.8 fmol X mm-1) were found in the thick ascending limb of the Henle's loop and in the collecting tubule, whereas specific binding could not be detected in the proximal tubule. In the medullary collecting tubule, the association and dissociation rate constants at 4 degrees C were k1 = 3.0 X 10(6) M-1 X min-1 and k-1 = 0.021 min -1; the ratio k-1/k1 = 7.0 nM was in agreement with the estimated equilibrium dissociation constant (Kd = 2.4 nM). [3H]PK 11195 binding sites from medullary ascending limb and medullary collecting tubule revealed the following sequence of specificity: PK 11195 = Ro 5-4864 much greater than clonazepam, indicating that tubule binding sites might be the peripheral benzodiazepine receptors of the rat kidney.     

Phosphatidylcholine Biosynthesis in the Neuroblastoma-Glioma Hybrid Cell Line NG 108-15: Stimulation by Phorbol Esters

Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High-affinity binding sites (KD = 2 nM for 1-h incubation at 20 degrees C) and low-affinity binding sites (KD = 40 nM) for 125I-labeled alpha-bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high-affinity binding of toxin is 0.56 nM (k1 = 6.3 X 10(-3) min-1 nM-1; k-1 = 3.5 X 10(-3) min-1) at 20 degrees C. Nicotine, d-tubocurarine, and acetylcholine are among the most effective inhibitors of high-affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulfhydryl residues. High-affinity toxin binding sites that have been subjected to reaction with the sulfhydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High-affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues.     

Specific ganglioside binding to receptor sites on T lymphocytes that couple to ganglioside-induced decrease of CD4 expression

The binding of different gangliosides to rat T-helper lymphocytes was characterized under conditions that decrease CD4 expression on different mammalian T-helper lymphocytes. Saturation binding by monosialylated [3H]-GM1 to rat T-lymphocytes was time- and temperature-dependent, had a dissociation constant (KD) of 2.2 +/- 1.4 microM and a binding capacity near 2 fmoles/cell. Competitive inhibition of [3H]-GM1 binding demonstrated a structural-activity related to the number of unconstrained sialic acid moieties on GM1-congeneric gangliosides. A comparison between the results of these binding studies and ganglioside-induced decrease of CD4 expression demonstrated that every aspect of [3H]-GM1 binding concurs with ganglioside modulation of CD4 expression. It is concluded that the specific decrease of CD4 expression induced by pretreatment with gangliosides involves the initial process of ganglioside binding to specific sites on CD4+ T-helper lymphocytes.     

Positive cooperativity of ryanodine binding to the calcium release channel of sarcoplasmic reticulum from heart and skeletal muscle

Ryanodine is a specific ligand for the calcium release channel which mediates calcium release in excitation-contraction coupling in muscle. In this study, ryanodine binding in sarcoplasmic reticulum from heart muscle and skeletal muscle is further compared and correlated with function. The new findings include the following: (1) Two types of binding, high affinity (KD1 approximately 5-10 nM) and low affinity (KD2 approximately 3 microM), can now be discerned for the skeletal muscle receptor. KD1 is approximately the same as and KD2 of similar magnitude to that previously reported for heart. (2) The dissociation rates for the high-affinity binding have been directly measured for both heart and skeletal muscle (t1/2 approximately 30-40 min). These rates are more rapid than previously reported (t1/2 approximately 14 h). (3) KD1's obtained from the ratio of the dissociation and association rate constants agree with the dissociation constant measured by equilibrium binding Scatchard analysis. (4) Ryanodine binding to the low-affinity site can be correlated with a decrease in the dissociation rate constant (k-1) of the high-affinity site, and thereby in the apparent dissociation constant (KD1). The inhibition constant (KI) for inhibiting the high-affinity off rate obtained from a double-reciprocal plot of the change in off rate vs [ryanodine] is practically the same in heart (0.66 microM) and skeletal muscle (0.64 microM) and in the range of the KD2. The binding of cold ryanodine to the low-affinity site appears to lock the bound [3H]ryanodine onto the high-affinity site rather than to exchange with it. Thus, in this sense, the ryanodine receptor exhibits "positive cooperativity".(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of Muscarinic Receptors in Rat Cerebral Cortical Microvessels

Microvessels isolated from rat cerebral cortex consist mainly of capillaries (greater than 85%). Fresh, intact microvessel preparations have been analyzed by radioligand binding techniques for muscarinic receptors. Scatchard analysis of specific quinuclidinyl benzilate (QNB) binding indicates that microvessels possess a large number of muscarinic sites (914 fmol/mg protein) of high affinity (KD = 0.034 nM). The association and dissociation rate constants (0.37 min-1 nM-1 and 0.0067 min-1, respectively) yield an equilibrium KD of 0.018 nM. Displacement of [3H]QNB by muscarinic ligands and control substances is typical of muscarinic receptors. The results indicate that cerebral microvessels possess a large population of muscarinic receptors.     

Thyroid hormone regulation of beta-adrenergic receptor number.

The effects of exogenous thyroid hormones (thyroxine and triiodothyronine) on beta-adrenergic receptors in the rat myocardium were investigated. The potent beta-adrenergic antagonist, (-)-[3H]dihydroalprenolol, was used to directly estimate the number and affinity of beta-adrenergic receptors in rat heart membranes from control and hyperthyroid rats. Cardiac membranes from hyperthyroid rats contained 196 +/- 7 fmol of (-)-[3H]dihydroalprenolol binding sites/mg of protein which was significantly (p less than 0.005) greater than the number of binding sites (89 +/- 5 fmol/mg of protein) present in control membranes. The equilibrium dissociation constant (KD) for the interaction of receptors with dihydroalprenolol was the same (2 to 15 nM) in membranes from control and hyperthyroid rats. Similarly, there was no significant difference between the control and hyperthyroid membranes in the affinity of the beta-adrenergic receptor binding sites for the beta-adrenergic agonist isoproterenol. The results of this study demonstrate that thyroid hormones can regulate the number of cardiac beta-adrenergic receptors. The increased numbers of receptors may be responsible, at least in part, for the enhanced catecholamine sensitivity of beta-adrenergic-coupled cardiac responses in the hyperthyroid state.     

Photoaffinity Labeling of the 5-HydroxytryptamineIA Receptor in Rat Hippocampus

1-[2-(4-Azidophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (p-azido-PAPP) inhibits [3H]5-hydroxytryptamine [( 3H]5-HT) binding to 5-HT1A and 5-HT1B sites in rat brain with equilibrium dissociation constants (KD) of 0.9 nM and 230 nM, respectively. [3H]p-Azido-PAPP was synthesized and its reversible and irreversible binding properties to the hippocampal 5-HT1A site characterized. [3H]p-Azido-PAPP labeled a single class of sites in rat hippocampal membranes with a KD of 1 nM and a maximal binding density of 370 fmol/mg protein. The pharmacological profile of [3H]p-azido-PAPP binding was consistent with the radioligand's selective interaction with the 5-HT1A receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes preincubated with [3H]p-azido-PAPP and irradiated showed a major band of incorporation of radioactivity at approximately 55,000 daltons. This incorporation could be blocked when membranes were incubated with 1 microM of several agents that have high affinity for 5-HT1A sites [5-HT, 8-hydroxy-2-(di-n-propylamino)tetraline, TVX Q 7821, spiperone, buspirone, d-lysergic acid diethylamide, metergoline]. The results indicate that on photolysis [3H]p-azido-PAPP irreversibly labels a polypeptide that is, or is a subunit of, the 5-HT1A receptor in rat hippocampus.     

The (--)[3H]dihydroalprenolol binding to rat adipocyte membranes: an explanation of curvilinear Scatchard plots and implications for quantitation of beta-adrenergic sites

In rat adipocyte membranes, both beta-adrenergic agonists and beta-adrenergic antagonists competed with (--)[3H]dihydroalprenolol for high affinity (KD 2-4 nM) and low capacity binding sites. The antagonists but not the agonists competed with (--)[3H]dihydroalprenolol for lower affinity and higher capacity sites. The present studies were performed in order to characterize the adipocyte beta-adrenergic receptor and distinguish it from low affinity, higher capacity sites which were heat-labile and not stereoselective. When isoproterenol was used to define the nonspecific binding, saturation studies showed a single binding site with a capacity of approximately 100 fmol/mg membrane protein (corresponding to approximately 50,000 sites/adipocyte). Binding was saturated by 10 nM (--)[3H]dihydroalprenolol. Approximate KD's of 204 nM were observed. Kinetic analysis of (--)[3H]dihydroalprenolol binding provided an independent measurement of KD between 0.75 and 1.1 nM. This binding site had the characteristics of a beta 1-adrenergic receptor with the potency of isoproterenol greater than norepinephrine greater than or equal to epinephrine as competitors of binding. Furthermore, the KD of inhibition of (--)[3H]dihydroalprenolol binding correlated with the Ki of inhibition by antagonists or Ka of activation by agonists of glycerol release in isolated adipocytes (r = 0.968, P less than 0.001). These results suggest that beta-adrenergic agonists compete with (--)[3H]dihydroalprenolol for the high affinity binding site which represents the physiological site. Furthermore, the use of antagonists (propranolol, alprenolol) to define specific beta-binding includes nonspecific site(s) as well as the beta-adrenergic site. Previous characterization and quantitation of beta receptors in rat fat cell membranes may have been in error by incorporating both types of binding in their measurement.     

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)     

Effect of a pentosan polysulphate upon thrombin and factor Xa inactivation by antithrombin III.

The kinetics of inhibition of human and bovine alpha-thrombin and human factor Xa by antithrombin III were examined under pseudo-first-order conditions as a function of the concentration of pentosan polysulphate [a fully sulphated (beta 1-4)-linked D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid]. Double-reciprocal plots of the observed first-order rate constant against concentration of pentosan polysulphate gave straight lines, intercepts on the axes giving values for maximum increase in second-order rate constant (by calculation) and apparent dissociation constant. These values were: for human alpha-thrombin 1.52 X 10(7) M-1 . min-1 and 3.6 microM respectively, for bovine alpha-thrombin 6.56 X 10(6) M-1 . min-1 and 0.16 microM and for factor Xa 6.86 X 106 M-1 . min-1 and 20 microM. In the presence of pentosan polysulphate the dissociation constant for the initial complex of antithrombin III and thrombin was shown to be reduced from approx. 2 X 10(-3) M to 61 X 10(-6) M without apparent change in the limiting rate constant of 750 min-1. An oligosaccharide (primarily 8-10 saccharide units) prepared from heparin and with high affinity for antithrombin III but low potency in the thrombin-antithrombin III interaction did not diminish the rate of interaction catalysed by pentosan polysulphate. The catalysis was shown to be due to a weak electrostatic interaction, since it was completely reversed by concentrations of NaCl greater than 0.3 M. It is concluded that the mechanism is independent of the heparin high-affinity binding site on antithrombin III and is probably due to binding of the high-charge-density polysaccharide to the proteinase. It is calculated that the acceleration in rate achieved, although lower than that of heparin, approaches that required to be of physiological significance and may be of importance in the anticoagulation role of antithrombin III at sites of high charge density which may occur in vivo.