Number Of High-affinity Sites Research Articles

Effects of the presence of Fe(0) on the sorption of lanthanum and lutetium mixtures in smectites

The sorption of La and Lu mixtures was examined in two bentonites after incubation for three months at 20 and 80°C with Fe(0), as a laboratory approach to evaluate the effects of waste canister corrosion in a deep repository on the performance of clay engineered barriers. The sorption/desorption parameters were determined from batch tests in two ionic media: deionized water and, to consider the additional effect of cement leachates, 0.02molL−1 Ca.Results from XRD analyses showed the formation of crystalline FeO(OH), goethite, in a few samples and the degradation of the bentonites due to Fe(0) oxidation during incubation. Moreover, the EDX spectra showed that the lanthanides were sorbed primarily at smectite sites, although sorption onto goethite was also observed, whereas Fe(0) particles did not contribute to lanthanide sorption. The formation of goethite could explain the high Kd values measured in a few scenarios (e.g., those with single solutions or mixtures with the lowest initial concentration of the competitive lanthanide in which high affinity sites governed sorption), with up to 3-fold increases over the values obtained without Fe incubation. However, at higher lanthanide concentration, Kd values decreased or remained constant compared to the samples without Fe incubation, which could be explained by bentonite degradation. In the Ca medium, as much as 5 times lower Kd values were obtained, because of the competitive effect of the Ca ions, especially for Lu in the MX80 bentonite. This indicated that the small number of high affinity sites had been diminished.The sorption data were satisfactorily fitted to a two-solute Langmuir model. In addition, Kd values correlated well with desorption data, which showed that the larger the decrease in Kd, the larger the increase in sorption reversibility. It is suggested that corrosion products from the metal canister might compromise the long-term radionuclide retention of the clay-engineered barriers.

Why Is α4β2 Nachr More Sensitive to Volatile Anesthetics Than α7 Nachr?

Two subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) show different functional sensitivities to volatile anesthetics: the α4β2 nAChR is hypersensitive while the α7 nAChR is insensitive. To understand why these homologous proteins have different functional responses to volatile anesthetics, we performed multiple sets of 20-ns molecular dynamics (MD) simulations on the closed- and open-channel α7 in the absence and presence of halothane, and compared the results with those from a similar study on the α4β2 nAChR (Liu, et al. 2009). The details about construction of receptor structural models were published (Haddadian, et al. 2008). Initial halothane docking and subsequent MD simulations revealed several halothane-binding sites in α7. Consistent with observations from α4β2, free energy perturbation calculations showed that halothane had higher binding affinity in the closed- than the open-channel; α7 and α4β2 had a comparable number of high affinity sites. GNM analysis showed that halothane induced profound changes in correlated domain motion of the open-channel α4β2, especially between the Cys-loop and the TM2-TM3 linker, but had a negligible impact to the motion of the α7. Salt bridges between these loops in the β2 subunit may be responsible for the aforementioned observation. Flexibility of several key loops in the open-channel α4β2 changed considerably in the presence of halothane, but the same loops in the open-channel α7 showed little change. Taken together, our results suggest that halothane binding in nAChRs may be necessary, but not sufficient to produce essential dynamical changes that alter protein functions. Although α7 and α4β2 are homologous, specific residues in key loops may make α4β2 more susceptible to volatile anesthetics while α7 unaffected. Supported by NIH (R01GM66358, R01GM56257, and T32GM075770) and NCSA through the PSC.

Chain Length-dependent Binding of Fatty Acid Anions to Human Serum Albumin Studied by Site-directed Mutagenesis

Human serum albumin is the most abundant protein in the circulatory system, and one of its principal functions is to transport fatty acids. Binding of octanoate, decanoate, laurate and myristate was studied by a rate-of-dialysis technique. The primary association constants increased, but not linearly, with chain length. The number of high-affinity sites also increased with chain length; octanoate and decanoate bind to one such site, whereas laurate and myristate most probably bind to two sites. Albumin is composed of three homologous helical domains (I–III), which can be subdivided into two subdomains (A and B). For getting information about the positions of the high-affinity sites we produced 13 recombinant isoforms mutated in four different subdomains. Results obtained with these albumins are in accordance with the following model: octanoate and decanoate bind to a single site in subdomain IIIA, laurate binds to sites in subdomains IIIA and IIIB, whereas myristate binds in subdomains IB and IIIB. The results also showed that primary fatty acid binding is sensitive to amino acid substitutions in other parts of the protein. This is in contrast to the effect of amino acid substitutions of genetic albumin variants (alloalbumins). Usually these substitutions, which are situated at the surface of the protein, have no effect on fatty acid binding. Binding of fatty acid anions to different high-affinity sites and the sensitivity of these sites to amino acid substitutions elsewhere in the protein (and perhaps also to other types of modifications) are important factors that could effect simultaneous binding of other ligands, e.g. in patients treated with albumin-binding drugs.

Adenosine transport in peripheral blood lymphocytes from Lesch-Nyhan patients.

We postulated that adenosine function could be related to some of the neurological features of Lesch-Nyhan syndrome and therefore characterized adenosine transport in PBLs (peripheral blood lymphocytes) obtained from Lesch-Nyhan patients (PBL(LN)) and from controls (PBL(C)). Adenosine transport was significantly lower in PBL(LN) when compared with that in PBL(C) and a significantly lower number of high affinity sites for [(3)H]nitrobenzylthioinosine binding were quantified per cell ( B (max)) in PBL(LN) when compared with that in PBL(C). After incubation with 25 microM hypoxanthine, adenosine transport was significantly decreased in PBL(LN) with respect to PBL(C). Hypoxanthine incubation lowers [(3)H]nitrobenzylthioinosine binding in PBL(C), with respect to basal conditions, but does not affect it in PBL(LN). This indicates that hypoxanthine affects adenosine transport in control and hypoxanthine-guanine phosphoribosyltransferase-deficient cells by different mechanisms.

The Cytoplasmic and Transmembrane Domains of the p75 and Trk A Receptors Regulate High Affinity Binding to Nerve Growth Factor

Ligand-induced receptor oligomerization is an established mechanism for receptor-tyrosine kinase activation. However, numerous receptor-tyrosine kinases are expressed in multicomponent complexes with other receptors that may signal independently or alter the binding characteristics of the receptor-tyrosine kinase. Nerve growth factor (NGF) interacts with two structurally unrelated receptors, the Trk A receptor-tyrosine kinase and p75, a tumor necrosis factor receptor family member. Each receptor binds independently to NGF with predominantly low affinity (K(d) = 10(-9) m), but they produce high affinity binding sites (K(d) = 10(-11) m) upon receptor co-expression. Here we provide evidence that the number of high affinity sites is regulated by the ratio of the two receptors and by specific domains of Trk A and p75. Co-expression of Trk A containing mutant transmembrane or cytoplasmic domains with p75 yielded reduced numbers of high affinity binding sites. Similarly, co-expression of mutant p75 containing altered transmembrane and cytoplasmic domains with Trk A also resulted in predominantly low affinity binding sites. Surprisingly, extracellular domain mutations of p75 that abolished NGF binding still generated high affinity binding with Trk A. These results indicate that the transmembrane and cytoplasmic domains of Trk A and p75 are responsible for high affinity site formation and suggest that p75 alters the conformation of Trk A to generate high affinity NGF binding.

Light Scatter and DNA Accessibility to Propidium Iodide of Ataxia Telangiectasia and Fanconi Anemia Cells

Cells from individuals with genetic diseases ataxia telangiectasia (AT) and Fanconi anemia (FA) exhibit hypersensitivity to ionizing radiation (AT) or DNA cross-linking agents (FA) which may be caused by multiple factors including defects in chromatin structure and DNA repair. In this study, a combination of cytometric techniques was employed to study the chromatin conformation of AT and FA cells. Nuclei of peripheral blood mononuclear cells (PBMCs) and of skin fibroblasts established from AT and FA patients were analyzed by light scattering and fluorimetric titration with the DNA-intercalating dye propidium iodide. The light scatter measurements revealed the presence of small-sized nuclei with reduced granularity in PBMCs and fibroblasts from both AT and FA patients. The fluorometric titration data could be interpreted by assuming two classes of propidium iodide binding sites with different affinities. The number of high-affinity sites in AT and FA fibroblasts was significantly larger (by 20%) than in control cells. Our findings show the applicability of cytometric techniques for the rapid assessment of chromatin conformation and also suggest the possibility to identify AT and FA carriers.

Uranium(VI) complexation with citric, humic and fulvic acids

The binding of uranium(VI) by Suwannee River humic and fulvic acids was studied at pH values of 4.0 and 5.0 in 0.10 M NaClO4using an ion-exchange technique. Few data sets currently exist for metal binding to different molecular weight fractions from the same source. The complexation of U(VI) by citric acid was also studied under the same experimental conditions in order to "calibrate" the experimental and modeling approaches. For the citric acid system, the experimental results were analyzed using Schubert´s ion-exchange method, which indicated the formation of only a 1:1 uranyl-citrate complex. Close agreement was found for the values of log β1,1(6.69±0.03 at I = 0.10) determined from nonlinear regression of data collected at pH values of 4.0 and 5.0. This value represents a more direct measurement of the binding constant for the 1:1 uranyl-citrate complex than do other existing literature values derived from experimental data requiring the simultaneous consideration of 1:1 and 2:2 species.Both humic and fulvic acids were demonstrated to strongly bind U(VI), with humic acid forming slightly stronger complexes and exhibiting greater pH dependence. Analyses of the data for the humic and fulvic acid systems using the Schubert´s equation previously applied to the citrate system result in an apparent nonintegral number of ligands binding the uranyl ion. Schubert´s method is only appropriate for interpreting mononuclear complexes with integral moles of binding ligands. Thus, a more elaborate binding model was required and the data were interpreted assuming either: (1) a mixture of 1:1 and 1:2 uranyl-ligand complexes or (2) a limited number of high affinity sites forming a 1:1 complex. While both of these modeling approaches are shown to provide excellent fits to the data, the second is deemed more appropriate given the large size of humic and fulvic acid molecules as well as previous results obtained with other metal cations, such as Cu(II).

Influence of Nonlinear Sorption Kinetics on the Slow-Desorbing Organic Contaminant Fraction in Soil

Release rates of hydrophobic organic compounds (HOCs) from the soil matrix influence the availability of HOCs in soils or sediments for microbial degradation or removal by physical means (e.g., soil washing or soil venting). In this study it was shown that the initial contaminant concentration influences the desorption rate. This was attributed to the presence of a limited number of high affinity sites that cause nonlinear sorption behavior. The experimental results could be described with a kinetic model composed of two separate compartments. One compartment was described with a Freundlich isotherm and corresponding kinetics and was assumed to represent sorption to high affinity sites. The second compartment was described with a linear sorption isotherm and first-order kinetics. The model was used to simulate the influence of purging strategies on removal of QCB. The simulations showed that after removal of a fast-desorbing fraction, the slow-desorbing fraction could be efficiently removed at very low purging rates. Intermittent purging reduced the total purging time but the simulations showed large fluctuations in the aqueous pentachlorobenzene concentration. For each subsequent purging interval, the purging efficiency decreased due to the nonlinear desorption kinetics of the slow-desorbing fraction of pentachlorobenzene.

Expression of the cardiac ryanodine receptor in the compensated phase of hypertrophy in rat heart

Abnormal calcium handling is a general feature of cardiac hypertrophy and alteration in the expression of SR proteins has been suggested to be involved in this alteration. To determine the expression of the cardiac ryanodine receptor (Ry2) gene during compensatory hypertrophy, we studied the mRNA and protein accumulation in left ventricles from rats with 30 to 100% hypertrophy. Cardiac hypertrophy was obtained after 1 month of aortic constriction. Ry2 mRNA was analyzed by RNase protection assay, Northern and slot blots, and Ry2 protein by high-affinity [3H]ryanodine binding and Western blot. We demonstrate that: (1) the cardiac Ry2 mRNA concentration is decreased by 50% in severe hypertrophy; (2) both the density of the high-affinity sites and the Ry2 protein level are decreased by 25%; (3) the decrease in the mRNA and protein levels and the number of high-affinity sites are highly correlated to the severity of hypertrophy. Our results suggest that, as for SR Ca(2+)-ATPase, there is either a downregulation or a lack of upregulation of the gene coding for the Ry2 in compensatory hypertrophy. The decreased density of Ry2 may alter SR Ca2+ transport and contribute to the impaired Ca2+ handling by slowing the Ca2+ movements.

Expression of the cholera toxin B subunit in the Golgi apparatus of Swiss 3T3 cells inhibits DNA synthesis induced by basic fibroblast growth factor.

We attempted to express the cholera toxin B subunit (CTXB) in the Golgi apparatus of cultured mammalian cells by means of gene transfection. Complementary DNA of CTXB was ligated with the Golgi-retention signal sequence of human beta 1,4 galactosyltransferase cDNA, and the chimeric gene yielded was inserted into a mammalian expression vector. The resultant construct was transfected into COS-1 cells for transient expression and into Swiss 3T3 cells for stable expression. The expression of a fusion protein encoded by the chimeric gene was demonstrated according to the following criteria: first, detection of a protein exhibiting the expected molecular mass on Western blot analysis using an anti-CTXB antibody; second, detection of the protein located in the Golgi area by indirect immunofluoresence microscopy; and third, detection of GM1 binding activity in cell lysates. Stable transformants satisfying the above criteria were subjected to an assay for mitogen-induced DNA synthesis. These transformants exhibited significantly lower DNA synthesis than mock transfection cells on stimulation with basic fibroblast growth factor (bFGF), whereas the two types of cells exhibited similar responses to 10% fetal calf serum and other mitogens, such as epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and platelet-derived growth factor. Analysis of the binding of radio-iodinated bFGF to the cells revealed that the transformants did not exhibit a significant decrease in the binding affinity or the number of high affinity sites. These results suggest that the fusion protein specifically inhibits the bFGF signaling not at the binding step but rather at a later step(s) triggered by the binding.

Differential subcellular localization of two populations of glutamate/AMPA receptors in the rat telencephalon

The distribution of glutamate AMPA receptors in the synaptosomal and microsomal fractions of neonatal and adult rat telencephalon was studied by determining the saturation kinetics at equilibrium of 3H-AMPA and 3H-CNQX binding. At both ages, synaptosomal preparations exhibited two populations of 3H-AMPA binding sites with a small number of high affinity sites and a large number of low affinity sites. 3H-AMPA binding to microsomal preparations from both neonatal and adult rat telencephalon exhibited a much higher proportion of high affinity relative to low affinity sites. 3H-CNQX binding to the same fractions did not parallel 3H-AMPA binding, but was correlated with the low affinity 3H-AMPA binding and with a marker of plasma membranes. The results suggest that nonsynaptic glutamate/AMPA receptors have a high affinity for agonist and become low affinity when inserted into postsynaptic membranes and that 3H-CNQX binds synaptic but not nonsynaptic glutamate/AMPA receptors with high affinity.

Modified high-affinity binding of Ni2+, Ca2+ and Zn2+ to natural mutants of human serum albumin and proalbumin

High-affinity binding of radioactive Ni2+, Ca2+ and Zn2+ to six genetic albumin variants and to normal albumin isolated from the same heterozygote carriers was studied by equilibrium dialysis at pH 7.4. The three cations bind differently to albumin. Ni2+ binds to a site in the N-terminal region of the protein which is partially blocked by the presence of a propeptide as in proalbumin (proAlb) Varese (Arg-2-->His), proAlb Christchurch (Arg-1-->Gln) and proAlb Blenheim (Asp1-->Val) and by the presence of only an extra Arg residue (Arg-1) as in Arg-Alb and albumin (Alb) Redhill. The association constants are decreased by more than one order of magnitude in these cases, suggesting biological consequences for the ligand. The additional structural changes in Alb Redhill have no effect on Ni2+ binding. Finally, the modification of Alb Blenheim (Asp1-->Val) reduces the binding constant to 50%. Ca2+ binding is decreased to about 60-80% by the presence of a propeptide and the mutation Asp1-->Val. Arg-1 alone does not affect binding, whereas Alb Redhill binds Ca2+ more strongly than the normal protein (125%). In contrast with binding of Ni2+ and Ca2+, albumin shows heterogeneity with regard to binding of Zn2+, i.e. the number of high-affinity sites was calculated to be, on average, 0.43. The binding constant for Zn2+ is increased to 125% in the case of proAlb Varese, decreased to 50-60% for proAlb Christchurch and Alb Redhill but is normal for proAlb Blenheim, Alb Blenheim and Arg-Alb. The effects of the mutations on binding of Ca2+ and Zn2+ indicate that primary binding, when operative, is to as yet unidentified sites in domain I of the albumin molecule.

Specific binding of the peroxisomal protein targeting sequence to glyoxysomal membranes.

It has been demonstrated that the carboxyl-terminal amino acid sequence, serine-lysine-leucine (SKL), is sufficient to direct a polypeptide to peroxisomes in vivo, and that this sequence is functional in plants, animals, and yeasts. Furthermore, many peroxisomal proteins have SKL carboxyl termini, including rat acyl-CoA oxidase. We have synthesized a 125I-peptide with the sequence of the last 12 amino acids of acyl-CoA oxidase, D-Tyr-HKHLKPLQSKL (SKLp), and used it to detect a receptor that recognizes SKL containing proteins targeted to glyoxysomes. SKLp binding to alkali-stripped glyoxysomal membranes was saturable and 80% of the binding could be displaced by 1 microM unlabeled SKLp or 8 micrograms/ml glyoxysomal matrix proteins. Very little specific binding was associated with endoplasmic reticulum or mitochondrial membranes. Specific binding was affected by the ionic composition of the medium; the binding was optimal at pH 6.5 and was inhibited by mono- and divalent cations. Scatchard analysis of SKLp binding to glyoxysomal membranes indicated that there were two binding sites with Kd values of 160 and 1450 nM and abundances of 17 and 43 nmol/mg glyoxysomal membrane protein, respectively. Protease treatment of the alkali-stripped glyoxysomal membranes lowered the number of high affinity sites and destroyed all the low affinity sites. These results demonstrate, for the first time, that there is an integral membrane protein in glyoxysomes that has the characteristics of a receptor for protein import.

Low density lipoprotein receptors bind and mediate cellular catabolism of normal very low density lipoproteins in vitro.

Very low density lipoproteins (VLDL) are heterogeneous, triglyceride-rich particles that are precursors of low density lipoproteins (LDL). Before conversion to LDL, the majority of VLDL are irreversibly cleared from plasma by uncertain mechanisms. To investigate one potential mechanism for VLDL clearance, we studied the ability of LDL receptors to mediate VLDL uptake in vitro. Small, intermediate, and large VLDL from normolipidemic humans were found to bind and undergo catabolism via LDL receptors on normal human fibroblasts. Binding to cell surfaces was up-regulated by lovastatin, an inducer of LDL receptors. Both LDL and a monoclonal antibody against the LDL receptor (IgG-C7) prevented binding of 125I-VLDL. Also, VLDL binding to mutant fibroblasts lacking LDL receptors was low. Thus, LDL receptors mediated VLDL interactions with cells. Binding affinity decreased near saturation, and the apparent number of high affinity sites decreased with increasing VLDL particle size. Because LDL receptors are small (M(r) 115,000) relative to VLDL (M(r) 9-24 x 10(6)) and are clustered in clathrin-coated pits, these findings suggest that steric hindrance becomes an important binding determinant near saturation and are consistent with a lattice model for LDL receptor-ligand interactions. The capacity for cellular catabolism of VLDL decreased with increasing particle size, consistent with a lattice model. The lattice model was also supported by differences between 125I-VLDL binding to cell surfaces and binding to partially purified LDL receptors in solid-phase assays in which steric constraints resulting from clustering in clathrin-coated pits are not present. In both cell-surface and solid-phase assays, VLDL bound via apoE, not apoB-100. Our studies establish that normal VLDL interact with LDL receptors and that steric hindrance due to crowding of particles on clustered LDL receptors is an important determinant of their binding and catabolism. These findings suggest that LDL receptors may participate in normal VLDL clearance in vivo.

Selective masking of M1-receptors in calf retina membranes by the venom of the marine snail Conus tessulatus.

The non discriminatory antagonist [3H]QNB labels M1- and M2-muscarinic receptors in calf retina membranes. Crude venom from the marine gastropod Conus tessulatus produces a partial decrease in [3H]QNB binding. The total number of sites (560 +/- 13 fmol/mg protein in control experiments) decreases to 370 +/- 10 fmol/mg protein whereas the affinity of the radioligand is unaffected (KD = 0.42 +/- 0.01 nM and 0.46 +/- 0.02 nM, respectively). This process is venom concentration-dependent, quasi-irreversible, and calcium-dependent. Proteolytic activity can not be detected. The partial effect of the venom is related to preferential masking of the M1-receptors. Competition curves of the M1-selective antagonist pirenzepine are shallow in control experiments: 45% of the receptors are of the M1-type (Ki = 45 +/- 6 nM) while the remaining are of the M2-type (Ki = 1.0 +/- 0.2 microM). In venom-treated membranes, only a low affinity site (M2-receptors, Ki = 1.5 +/- 0.4 microM) is detected by pirenzepine competition binding. Saturation binding experiments reveal that the venom causes a substantial decrease in the number of high affinity sites for [3H]pirenzepine without affecting its KD (23 +/- 4 nM and 20 +/- 6 nM in control- and venom-treated membranes respectively). The venom produces a leftward shift of the carbachol/[3H]QNB competition binding curve, but the ability of 0.1 mM GTP to confer a rightward shift of the competition curve is not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Reciprocal modulation of the binding of angiotensin agonists and antagonists to angiotensin receptors in smooth muscle

1. 1. Direct ligand binding studies have shown that the agonist 125I-[Sar 1]Ang II and the antagonist 125I-[Sar 1Ile 8]Ang II bind to bovine uterus smooth muscle membranes in a time-dependent, reversible and saturable manner; both ligands had the same number of high affinity sites. 2. 2. [Sar 1Ile 8]Ang II inhibited the binding of 125I-[Sar 1]Ang II in a non-competitive manner by decreasing the number of high affinity sites without changing the binding affinity of the radioligand. 3. 3. [Sar 1]Ang II also inhibited the binding of 125I-[Sar 1Ile 8]Ang II in a non-competitive manner. 4. 4. Dissociation of both radioligands from their receptor sites was fast enough that pseudo irreversible occupancy of the binding sites could not account for the observed non-competitive inhibition. 5. 5. Displacement studies using 125I-[Sar 1Ile 8]Ang II as the radioligand provided evidence for the existence of two binding sites when the displacing ligand was [Sar 1]Ang II but not when the displacing ligand was [Sar 1Ile 8]Ang II. 6. 6. GTPSγS had no discernible effect on the binding of either 125I-[Sar 1]Ang II or 125I-[Sar 1Ile 8]Ang II to bovine uterine membranes. 7. 7. The present findings are consistent with an allosteric mechanism of antagonism for [Sar 1Ile 8]Ang II. The data are also consistent with a mechanism wherein agonist and antagonist ligands occupy different binding modes at the same receptor site and induce long-term conformational changes in the receptor which are idiosyncratic with respect to the nature of the ligand. An emerging relationship between the actions of angiotensin peptides and non-peptide mimetics of angiotensin is presented.

The cocaine analog WIN 35,428 binds to two sites in fresh rat caudate-putamen: Significance of assay procedures

Binding of the potent cocaine analog [ 3H]WIN 35,428 (2β-carbomethoxy-3β-(4-fluoro-phenyl)-tropane) was examined in membrane homogenates prepared from fresh rat caudate-putamen. The number of rinses of the membranes following incubation with the ligand was varied. After one wash, only a single binding site was evident. With an increasing number of washes, however, two binding sites were observed. Additional rinses, up to a total of four, led to a decrease in the number of low affinity binding sites, with no change in the number of high affinity sites. These results suggest that the methodology used in these binding assays may be important for interpretation of the results.

Plasminogen activator inhibitor-1 binds to fibrin and inhibits tissue-type plasminogen activator-mediated fibrin dissolution.

Plasminogen activator inhibitor-1 (PAI-1) accumulates within thrombi and forming whole blood clots. To explore this phenomenon at the molecular level, PAI-1 binding to fibrin was examined. The experiments were performed by adding 125I-PAI-1, which retains its complete tissue-type plasminogen (t-PA) inhibitory activity, to fibrin matrices formed in 2-cm2 tissue culture wells. Guanidine HCl-activated PAI-1 binding was reversible and was inhibited in the presence of excess, unlabeled PAI-1. Activated 125I-PAI-1 recognized 2 sites on fibrin: a very small number of high affinity sites (Kd less than 1 nM) and principally a large number of low affinity sites with an approximate Kd of 3.8 microM. Latent PAI-1 bound to fibrin at a site indistinguishable from the lower affinity site recognized by activated PAI-1. Fibrin, pretreated with activated PAI-1, was protected from t-PA-mediated plasmin degradation in a PAI-1 dose-responsive manner (IC50 = 12.3 nM). Clot protection correlated with partial occupancy of the low affinity PAI-1 binding site on fibrin and was due to the formation of sodium dodecyl sulfate-stable, PAI-1.t-PA complexes. Latent PAI-1 (27 nM) did not protect the fibrin from dissolution. The localization of PAI-1 to a thrombus by virtue of its fibrin binding potential could result in significant protection of the thrombus from the degradative effects of the fibrinolytic system.

"5-HT1R" or 5-HT1D sites? Evidence for 5-HT1D binding sites in rabbit brain.

Radioligand binding studies were performed in membranes of rabbit whole brain and striatum using the novel iodinated radioligand for 5-hydroxytryptamine 5-HT1B and 5-HT1D sites, Serotonin-5-O-Carboxymethyl-Glycyl[125I]Tyrosinamide ([125I]GTI). [125I]GTI labelled a finite number of high affinity sites in rabbit brain membranes, Bmax = 191 +/- 47 fmol/mg protein, pKD (-log mol/l) = 8.50 +/- 0.13, n = 5. The pharmacological profile of [125I]GTI binding was fully comparable to that reported previously in human and other brain preparations known to possess 5-HT1D sites (using either [3H]5-HT or [125I]GTI) and displayed a characteristic rank order of affinity: 5-carboxamido-tryptamine greater than 5-HT = dihydroergotamine greater than or equal to ergotamine greater than or equal to sumatriptan greater than or equal to CGS 12066 greater than or equal to metergoline greater than yohimbine greater than or equal to methysergide greater than ICYP greater than 8-OH-DPAT greater than or equal to CP 93129 greater than (-)pindolol greater than ketanserin greater than isamoltane greater than mesulergine greater than corynanthine greater than buspirone greater than MDL 72222. Autoradiographic studies were performed on rabbit brain slices using [3H]5-HT in the presence of 100 nmol/l 8-OH-DPAT and mesulergine (in order to mask 5-HT1A and 5-HT1C binding sites) and [125I]CYP (iodocyanopindolol) in the presence of 3 mumol/l isoprenaline and 100 nmol/l 8-OH-DPAT (in order to mask beta adrenoceptor and 5-HT1A binding sites). There was no detectable specific binding of [125I]CYP through the brain, thus excluding the presence of 5-HT1B sites in rabbit brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine receptors in human foetal brains: Characterization, regulation and ontogeny of [ 3H]spiperone binding sites in striatum

Eighteen corpora striata from normal human foetal brains ranging in gestational age from 16 to 40 weeks and five from post natal brains ranging from 23 days to 42 years were analysed for the ontogeny of dopamine receptors using [ 3H]spiperone as the ligand and 10 mM dopamine hydrochloride was used in blanks. Spiperone binding sites were characterized in a 40-week-old foetal brain to be dopamine receptors by the following criteria: (1) It was localized in a crude mitochondrial pellet that included synaptosomes; (2) binding was saturable at 0.8 nM concentration; (3) dopaminergic antagonists spiperone, haloperidol, pimozide, trifluperazine and chlorpromazine competed for the binding with IC 50 values in the range of 0.3–14 nM while agonists—apomorphine and dopamine gave IC 50 values of 2.5 and 10 μM, respectively suggesting a D 2 type receptor. Epinephrine and norepinephrine inhibited the binding much less efficiently while mianserin at 10 μM and serotonin at 1 mM concentration did not inhibit the binding. Bimolecular association and dissociation rate constants for the reversible binding were 5.7 × 10 8 M −1 min −1 and 5.0 × 10 −2 min −1, respectively. Equilibrium dissociation constant was 87 pM and the K D obtained by saturation binding was 73 pM. During the foetal age 16 to 40 weeks, the receptor concentration remained in the range of 38–60 fmol/mg protein or 570–1080 fmol/g striatum but it increased two-fold postnatally reaching a maximum at 5 years Significantly, at lower foetal ages (16–24 weeks) the [ 3H]spiperone binding sites exhibited a heterogeneity with a high ( K D, 13–85 pM) and a low ( K D, 1.2–4.6 nM) affinity component, the former accounting for 13–24% of the total binding sites. This heterogeneity persisted even when sulpiride was used as a displacer. The number of high affinity sites increased from 16 weeks to 24 weeks and after 28 weeks of gestation, all the binding sites showed only a single high affinity. GTP decreased the agonist affinity as observed by dopamine competition of [ 3H]spiperone binding in 20-week-old foetal striata and at all subsequent ages. GTP increased IC 50 values of dopamine 2 to 4.5 fold and Hill coefficients were also increased becoming closer to one suggesting that the dopamine receptor was susceptible to regulation from foetal life onwards.