Interaction of pirprofen enantiomers with human serum albumin.

The interaction of pirprofen enantiomers with human serum albumin (HSA) was investigated by means of high-performance liquid chromatography (HPLC), circular dichroism (CD), and 1H NMR spectroscopy. HPLC experiments indicated that both pirprofen enantiomers were bound to one class of high-affinity binding sites (n(+) = 1.91 +/- 0.13, K(+) = (4.09 +/- 0.64) x 10(5) M-1, n(-) = 2.07 +/- 0.13, K(-) = (6.56 +/- 1.35) x 10(5) M-1) together with nonspecific binding (n'K'(+) = (1.51 +/- 0.21) x 10(4) M-1, n'K'(-) = (0.88 +/- 0.13) x 10(-4) M-1). Slight stereoselectivity in specific binding was demonstrated by the difference in product n(+)K(+) = (0.77 +/- 0.08) x 10(6) M-1 vs. n(-)K(-) = (1.30 +/- 0.21) x 10(6) M-1, i.e., the ratio n(-)K(-)/n(+)K(+) = 1.7. CD measurements showed changes in the binding sites located on the aromatic amino acid side chains (a small positive band at 315 nm and a pronounced negative extrinsic Cotton effect in the region 250-280 nm). The protein remains, however, in its predominantly alpha-helical conformation. The 1H NMR difference spectra confirmed that both pirprofen enantiomers interacted with HSA specifically, most probably with site II on the albumin molecule.     

Characterization of the adenine binding sites of two Dolichos biflorus lectins.

The seed lectin and a stem and leaf lectin (DB58) from Dolichos biflorus have high-affinity hydrophobic sites that bind to adenine. The present study employs a centrifugal filtration assay to characterize these sites. The seed lectin contains two identical sites with Ka's of 7.31 x 10(5) L/mol whereas DB58 has a single site with a Ka of 1.07 x 10(6) L/mol. The relative affinities of these sites for a host of adenine analogs and derivatives were determined by competitive displacement assays. The most effective competitors for adenine were the cytokinins, a class of plant hormone, for which the lectins had apparent Ka's of 1.96 x 10(5)-4.90 x 10(4) L/mol. Direct binding of the cytokinin 6-(benzylamino)purine (BAP) to both lectins showed positive cooperativity for only the seed lectin, indicating the interaction of this ligand with more than one class of hydrophobic binding site. Fluorescence enhancement assays demonstrate cooperativity between hydrophobic sites of the seed lectin and also suggest that BAP binds to more than one class of site.     

The binding of palmitoyl-CoA to bovine serum albumin

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol of palmitoyl-CoA bound per mol of BSA (nu) versus -log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites (k1 = (1.55 +/- 0.46) x 10(-6) M-1) and four lower affinity secondary binding sites (k2 = (1.90 +/- 0.09) x 10(-8) M-1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmitoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K1 greater than K2 greater than K3 greater than...greater than or equal to K6), suggesting that each mol of long-chain acyl-CoA binds to BSA with decreasing affinities.     

Interaction of propafenone enantiomers with human alpha 1-acid glycoprotein

The interaction of propafenone enantiomers with human alpha 1-acid glycoprotein was studied using high-performance liquid chromatography. Each of the two optical antipodes interacted with one class of high-affinity binding sites characterized by Ka(R) = (6.18 +/- 0.93) x 10(5) M-1, n(R) = 1.34 +/- 0.09 for the (R)-isomer and Ka(S) = (8.93 +/- 1.82) x 10(5) M-1, n(S) = 0.99 +/- 0.08 for the (S)-isomer. Nonspecific binding to secondary low-affinity high-capacity binding site(s) was only slightly greater in the case of the (S)-enantiomer (n'k'(S) = (1.06 +/- 0.09) x 10(4) M-1) compared to the (R)-enantiomer (n'k'(R) = (6.87 +/- 0.72) x 10(3) M-1). It was concluded that both enantiomers interact with common single class of high-affinity binding sites on AAG (along with nonspecific binding) exhibiting only slight stereoselectivity for propafenone.     

Calpain 1 binding capacities of the N1-line region of titin are significantly enhanced by physiological concentrations of calcium

Calpain 1, an ubiquitous well-known calcium-dependent intracellular protease, was recently shown to bind tightly to the proximal end of the I-band titin segment in a calcium-dependent manner [Raynaud et al. (2005) FEBS J. 272, 2578-2590]. In the present work we identified the titin Ig-domain of concern by this interaction and the role of calcium in this interaction using a recombinant fragment of titin spanning the I2-I6 region and its subfragments. The heterodimeric form of calpain 1 binds to this titin fragment with a very high affinity ( K d = 5.1 +/- 0.2 x 10 (-7) M) at much lower calcium levels than those saturating the high-affinity binding sites of the peptidase ( K d = 25 microM). Investigation of this interaction with I2-I6 subfragments clearly showed that the dimeric form of calpain 1 binds exclusively to the Ig-domain I4 of titin with an affinity similar to that of the whole I2-I6 segment. As for the I2-I6 fragment, this interaction is calcium regulated. Calcium was shown to bind tightly to titin ( K d = 1.9 x 10 (-7) M), causing an oligomerization of the titin segment. At physiological calcium concentration (10 (-6) to 10 (-8) M), the prevailing form of the titin fragment is a trimer, suggesting that calpain 1 binds to this titin structure. From the present findings, it was concluded that calcium binding to titin increased the amount of bound calpain 1 (up to 40% of the total calpain 1) and that this bound calpain 1 might constitute a reservoir for this peptidase. In this context, we proposed a schematic diagram of this series of calcium-dependent events with the inherent unanswered questions. These events are probably under a complex regulation involving undoubtedly different yet unidentified proteins.     

Energetics of carbohydrate binding to Momordica charantia (bitter gourd) lectin: an isothermal titration calorimetric study

Physico-chemical and carbohydrate binding studies have been carried out on the Momordica charantia (bitter gourd) seed lectin (MCL). The lectin activity is maximal in the pH range 7.4-11.0, but decreases steeply below pH 7.0. The lectin activity is mostly unaffected in the temperature range 4-50 degrees C, but a sharp decrease is seen between 50 and 60 degrees C, which could be correlated to changes in the structure of the protein as seen by circular dichroism and fluorescence spectroscopy. Isothermal titration calorimetric studies show that the tetrameric MCL binds two sugar molecules and the binding constants (Kb), determined at 288.15 K, for various saccharides were found to vary between 7.3 x 10(3) and 1.52 x 10(4)M(-1). The binding reactions for all the saccharides investigated were essentially enthalpy driven, with the binding enthalpies (DeltaHb) at 288.15 K being in the range of -50.99 and -43.39 kJ mol(-1), whereas the contribution to the binding reaction from the entropy of binding was negative, with values of binding entropy (DeltaSb) ranging between -99.2 and -72.0 J mol(-1)K(-1) at 288.15 K. Changes in heat capacity (DeltaCp) for the binding of disaccharides, lactose and lactulose, were significantly larger in magnitude than those obtained for the monosaccharides, methyl-beta-D-galactopyranoside, and methyl-alpha-D-galactopyranoside, and could be correlated reasonably well with the surface areas of these ligands. Enthalpy-entropy compensation was observed for all the sugars studied, suggesting that water structure plays an important role in the overall binding reaction. CD spectroscopy indicates that carbohydrate binding does not lead to significant changes in the secondary and tertiary structures of MCL, suggesting that the carbohydrate binding sites on this lectin are mostly preformed.     

Enantioselective plasma protein binding of propafenone: mechanism, drug interaction, and species difference

The interaction of propafenone (PPF) enantiomers with human plasma, human serum albumin (HSA), alpha(1)-acid glycoprotein (AGP), as well as with plasma from rat, rabbit, and cow was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques. The stronger binding of the S-PPF found in human plasma was due to AGP. Two classes of binding sites in AGP were identified: one with high-affinity and small binding capacity (K(1(S)) = 7.65 x 10(6) M(-1), n(1(S)) = 0.50; K(1(R)) = 2.81 x 10(6) M(-1), n(1(R)) = 0.46), which revealed stereoselectivity; the other with low-affinity and high-binding capacity (n(2(S)) K(2(S)) = 9.95 x 10(3) M(-1); n(2(R)) K(2(R)) = 9.74 x 10(3) M(-1)). The binding to HSA was found to be weak and not enantioselective (nK(S) = 2.08 x 10(3) M(-1), nK(R) = 2.05 x 10(3) M(-1)). The interaction between enantiomers observed in human plasma was confirmed as a competitive type interacting at the high-affinity site in AGP. The binding mode of both enantiomers with AGP was mainly hydrophobic bond. PPF enantiomers had higher-binding affinity for the F-S variant of human AGP. Drug-drug binding interaction studies showed that verapamil, diazepam, nifedipine, furosemide, nitrendipine, and nimodipine did not affect the binding of PPF enantiomers except quinidine and aprindine at the therapeutic concentration. Comparative studies indicated considerable species-dependent binding stereoselectivity between plasma of the four species investigated.     

Anion transport in red blood cells. III. Sites and sidedness of inhibition by high-affinity reversible binding probes

Studies of binding of the reversible inhibitor DNDS (for abbreviations, see Nomenclature) and red blood cell membranes revealed 8.6 +/- 0.7 x 10(5) high-affinity binding sites per cell (KD = 0.8 +/- 0.4 muM). Under conditions of "mutual depletion," inhibition studies of anion exchange revealed 8.0 +/- 0.7 x 10(5) DNDS inhibitory sites per cell (KD = 0.87 +/- 0.04 muM). Binding and kinetics studies with DNDS indicate that there are 0.8 -- 0.9 x 10(6) functional anion transport sites per blood cell. The transport of DNDS displayed high temperature and concentration dependencies, chemical specificity, susceptibility to inhibition by DIDS, and differences between egress and ingress properties. Under conditions of no DNDS penetration (e.g., 0 degrees C), inhibition of anion exchange by DNDS showed marked sidedness from the outside inhibitions and were demonstrable at micromolar concentrations, whereas from the inside no inhibition occurred even at millimolar concentrations. The asymmetry of DNDS transport properties and the sidedness of binding and inhibition suggest that anion transport sites have a very low affinity for or are inaccessible to DNDS at the inner membrane face. The site of DNDS permeation, although susceptible to DIDS, is apparently not the site of anion exchange.     

Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus

Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.     

Dual effect of vasoactive intestinal peptide on the mitogenic response of rabbit spleen lymphocytes

The effects of vasoactive intestinal peptide (VIP) have been investigated on the mitogenic response of rabbit spleen cells. Specific binding of 125I-VIP to these mononuclear cells is rapid and saturable. Analysis of binding reveals two classes of binding sites, a class of high-affinity binding sites with KD = 0.93 +/- 0.11 nM and maximal binding capacity of 2000 +/- 560 sites/cell, and a class of low-affinity binding sites with KD = 225 +/- 58 nM and maximal binding capacity of 280,000 +/- 60,000 sites/cell. The VIP regulatory effect on mitogen-stimulated rabbit spleen cell proliferation appears to be time dependent and bimodal. When VIP was added simultaneously with mitogens, it induced an inhibition of the proliferative response. With concanavalin A (Con A) or pokeweed mitogen (PWM), addition of 10(-8) M VIP resulted in a maximal 30% inhibition of [3H]thymidine incorporation after 96 h of culture. This inhibitory effect was significant at concentrations from 10(-8)-10(-6) M and half-maximal inhibition was obtained with 1.2 x 10(-9) M VIP. By contrast, when rabbit spleen cells were preincubated for 18 h with VIP alone, the lymphocyte proliferative response to Con A was increased. However, this increase was mitogen-selective, since it was only observed when the T-cell mitogen Con A was used. The maximal response was obtained after 96 h of culture in the presence of Con A. The VIP stimulatory effect was dose-dependent with a maximal effect at 10(-7) M and a half-maximal effect at 1.7 x 10(-9) M VIP. The effect of VIP was also time-dependent, since a 6 h preincubation was sufficient to induce a significant increase in the proliferative response which was maximal after an 18 h preincubation.     

Carbohydrate-binding properties of L-fucose, D-mannose-specific lectin (SFL 100-2) particles produced by Streptomyces sp

L-Fucose, D-mannose-specific lectin (SFL 100-2) particles produced by Streptomyces no. 100-2 were labeled with N-succinimidyl-[2,3-3H]propionate to investigate quantitatively their binding properties to human erythrocytes. The labeling did not influence the physical properties or the hemagglutinating activity of the lectin particles. The binding studies suggested that two kinds of receptor sites were present on the erythrocytes. Association constants (Ka's) of the lectin particles to the receptor sites and the numbers of the receptor sites (n) on human O erythrocytes were calculated to be 4.60 X 10(8) M-1 and 3.17 X 10(4)/cell for high-affinity receptor sites, and 7.5 X 10(7) M-1 and 1.33 X 10(5)/cell for low-affinity ones. The inhibition constants (Ki's) for L-fucose, p-nitrophenyl (PNP)-beta-L-fucoside, D-mannose, and PNP-alpha-D-mannoside were calculated to be 1.20 X 10(3), 1.82 X 10(3), 1.82 X 10(2), and 2.40 X 10(2) M-1, respectively. The numbers of carbohydrate-binding sites (m) on the lectin particles were estimated to be 2.82, 2.18, 2.19, and 2.21 for L-fucose, PNP-beta-L-fucoside, D-mannose, and PNP-alpha-D-mannoside, respectively, suggesting that SFL 100-2 has two carbohydrate-binding sites per particle.     

Modulation of interleukin 2 high-affinity binding by lymphocyte-derived tetrahydrobiopterin: pterins as potential participants in the control of interleukin 2 receptor assembly

In this report, we have examined whether (6R)-tetrahydrobiopterin (H4biopterin) modulates the binding of interleukin 2 to high-affinity sites of the cloned mouse cytotoxic T-lymphocyte clone CTLL-2. Scatchard plot analysis of the equilibrium binding data reveals increased affinity when the cells are exposed simultaneously to interleukin 2 and to the pterin. The Kd values are statistically significantly reduced from 1.4 x 10(-11) M to 0.78 x 10(-11) M interleukin 2. The dissociation kinetics of the ligand were followed at 4 degrees C after equilibrium binding under high-affinity conditions (1.2 x 10(-10) M interleukin 2). In the presence of H4 biopterin, the dissociation rate constant (k-1) decreases from 6.2 x 10(-3) min-1 to 3.0 x 10(-3) min-1 and the half-time for dissociation increases from 106.8 min to 218.0 min. As a third approach interleukin 2 was bound to the surface of cells under high-affinity conditions by incubation in the cold and the internalization kinetics upon warming were determined. Sigmoidal-shaped kinetics of endocytosis in control cells indicate that the internalization rates increase only gradually. The presence of H4 biopterin causes an apparent immediate transition from higher-order kinetics to a linear response so that maximum internalization rates are reached immediately upon warming. The data show that lymphocyte-derived H4 biopterin in vitro at concentrations ranging from 2-8 x 10(-7) M modulates interleukin 2 high-affinity binding and that H4 biopterin potentially participates in the control of interleukin 2 receptor assembly.     

Thermodynamic studies on the interaction of water-soluble porphyrins with the glucose/mannose-specific lectin from garden pea (Pisum sativum)

Due to the application of porphyrins as photosensitizers in photodynamic therapy to treat cancer, and the ability of some lectins to preferentially recognize tumor cells, studies on the interaction of porphyrins with lectins are of considerable interest. Here we report thermodynamic studies on the interaction of several free-base and metallo-porphyrins with pea (Pisum sativum) lectin (PSL). Association constants (Ka) were obtained by absorption titrations by monitoring changes in the Soret band of the porphyrins and the Ka values obtained for various porphyrins at different temperatures are in the range of 1.0 x 10(4) to 8.0 x 10(4) M(-1). Both cationic and anionic porphyrins were found to bind to PSL with comparable affinity. Presence of 0.1 M methyl-alpha-D-mannopyranoside--a carbohydrate ligand that is specifically recognised by PSL--did not affect the binding significantly, suggesting that porphyrin and sugar bind at different sites on the lectin. From the temperature dependence of the Ka values, the thermodynamic parameters, change in enthalpy and change in entropy associated with the binding process were estimated. These values were found to be in the range: delthaH degree = -95.4 to -33.9 kJ x mol(-1) and deltaS degree = -237.2 to -32.2 J x mol(-1) x K(-1), indicating that porphyrin binding to pea lectin is driven largely by enthalpic forces with the entropic contribution being negative. Enthalpy-entropy compensation was observed in the interaction of different porphyrins to PSL, with the exception of meso-tetra-(4-sulfonatophenyl)porphyrinato zinc(II), emphasizing the role of water structure in the overall binding process. Circular dichroism and differential scanning calorimetric studies indicate that while porphyrin binding does not induce significant changes in the lectin structure and thermal stability, carbohydrate binding induces moderate changes in the tertiary structure of the protein and also increases its thermal unfolding temperature and the enthalpy of the unfolding transition.     

Selective distinction at equilibrium between the two alpha-neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration

The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.     

Comparative binding of wheat germ agglutinin and its succinylated form on lymphocytes

We have compared the binding parameters of native wheat germ agglutinin (WGA) and its succinylated form (SWGA) to rat lymphocytes. Scatchard plots were obtained with the fluoresceinated lectins in a concentration range of 10 nM to 0.1 mM. Association and dissociation rate parameters were also measured. The following differences were observed: at low concentration of WGA, binding is positively cooperative with a Hill coefficient of 1.75, whereas binding of SWGA is not. The numbers of high-affinity sites are respectively (2.5 +/- 0.8) X 10(6) and (6.4 +/- 1.3) X 10(5) for WGA and SWGA. Association constants were found to be (4.7 +/- 1.7) X 10(6) l mol-1 for WGA and (1.42 +/- 0.36) X 10(7) l mol-1 for SWGA, which is 35 times higher than for native WGA. Neuraminidase treatment decreases the Hill coefficient as well as the number of sites involved in the cooperative binding of native WGA. Equilibrium data were obtained at three temperatures to determine the thermodynamic parameters (delta H degree and delta S degree). These results are indicative of an oligomerization process dynamically formed at the membrane level before tight binding of the lectin to its receptors could occur.     

Ligand-induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells

Recently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X-100-insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady-state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF-binding sites: a high-affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 x 10(4) sites per cell) and a low-affinity site with a KD of 8.5 nM (1.9 x 10(6) sites per cell). Non-equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast-dissociating site, with a dissociation rate constant (k-1) of 1.1 x 10(-3) s-1 (1.0-1.3 x 10(6) sites per cell) and a slow-dissociating site, with a k-1 of 3.5 x 10(-5) s-1 (0.6-0.7 x 10(6) sites per cell). The cytoskeleton of A431 cells was isolated by Triton X-100 extraction. Scatchard analysis revealed that approximately 5% of the original number of receptors were associated with the cytoskeleton predominantly via high-affinity sites (KD = 1.5 nM). This class of receptors is further characterized by the presence of a fast-dissociating component (k-1 = 2.0 x 10(-3) s-1) and a slow-dissociating component (k-1 = 9.1 x 10(-5) s-1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4 degrees C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton-associated receptors appeared to represent low-affinity binding sites (KD = 7 nM). Dissociation kinetics also revealed an increase of fast-dissociating sites. These results indicate that at 4 degrees C EGF induces the binding of low-affinity, fast-dissociating sites to the cytoskeleton of A431 cells.     

Characterization of the binding sites for nimodipine and (-)-desmethoxyverapamil in bovine cardiac sarcolemma

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased thyrotropin binding in hyperfunctioning thyroid nodules

The object of this study was to investigate TSH receptors in hyperfunctioning thyroid nodules (HFN). In HFN, obtained from seven patients, 125-I-TSH binding as determined by equilibrium binding analysis on particulate membrane preparations, was found to be significantly increased as compared with normal thyroid tissues (five patients; P less than 0.001). Scatchard analysis of TSH-binding revealed two kinds of binding sites for both normal thyroid tissue and HFN, and displayed significantly increased association constants of high- and low-affinity binding sites in HFN (Ka = 11.75 +/- 6.8 10(9) M-1, P less than 0.001 and Ka = 2.1 +/- 1.0 10(7) M-1, P less than 0.025; x +/- SEM) as compared with normal thyroid tissue (Ka = 0.25 +/- 0.06 10(9) M-1, Ka = 0.14 +/- 0.03 10(7) M-1; x +/- SEM). The capacity of the high-affinity binding sites in HFN was found to be decreased (1.8 +/- 1.1 pmol/mg protein, x +/- SEM) in comparison with normal thyroid tissue (4.26 +/- 1.27 pmol/mg protein; x +/- SEM). TSH-receptor autoradiography applied to cryostatic tissue sections confirmed increased TSH binding of the follicular epithelium in HFN. These data suggest that an increased affinity of TSH-receptor sites in HFN in iodine deficient areas may be an important event in thyroid autonomy.     

Characterization of a putative SchistoFLRFamide receptor in the CNS of Locusta migratoria

A putative SchistoFLRFamide receptor in CNS membrane preparations of Locusta migratoria was characterized by cold competition binding and kinetic binding assays using [125I][Y(1)]SchistoFLRFamide ([125I]YDVDHVFLRFamide) as a radioligand. Binding to this site was saturable, specific, reversible, and of high-affinity. Data fit to a single-site binding model by non-linear regression (r(2) = 0.99) estimated K(d) = 1.73 +/- 0.45 x 10(-9) M and B(max) = 49.0 +/- 12.2 fmol.mg(-1) tissue. Total binding of [125I][Y(1)]SchistoFLRFamide to membrane preparations was reduced in the presence of GTPgammaS, an indication that the putative receptor is G protein-coupled. Structure-activity studies determined that the minimum sequence required for binding was HVFLRFamide. Other aspects of the ligand receptor interaction were also examined.     

Thermodynamics of the binding of galactopyranoside derivatives to the basic lectin from winged bean (Psophocarpus tetrogonolobus).

The thermodynamics of the binding of D-galactopyranoside (Gal), 2-acetamido-2-deoxygalactopyranoside (GalNAc), methyl-alpha-D-galactopyranoside, and methyl-beta-D-galactopyranoside to the basic agglutinin from winged bean (WBAI) in 0.02 M sodium phosphate and 0.15 M sodium chloride buffer have been investigated from 298.15 to 333.15 K by titration calorimetry and at the denaturation temperature by differential scanning calorimetry (DSC). WBAI is a dimer with two binding sites. The titration calorimetry yielded single-site binding constants ranging from 0.56 +/- 0.14 x 10(3) M-1 for Gal at 323.15 K to 7.2 +/- 0.5 x 10(3) M-1 for GalNAc at 298.15 K and binding enthalpies ranging from -28.0 +/- 2.0 kJ mol-1 for GalNAc at 298.15 K to -14.3 +/- 0.1 kJ mol-1 for methyl-beta-D-galactopyranoside at 322.65 K. The denaturation transition consisted of two overlapping peaks over the pH range 5.6-7.4. Fits of the differential scanning calorimetry data to a two-state transition model showed that the low temperature transition (341.6 +/- 0.4 K at pH 7.4) consisted of two domains unfolding as a single entity while the higher temperature transition (347.8 +/- 0.6 K at pH 7.4) is of the remaining WBAI dimer unfolding into two monomers. Both transitions shift to higher temperatures and higher calorimetric enthalpies with increase in added ligand concentration at pH 7.4. Analysis of the temperature increase as a function of added ligand concentration suggests that one ligand binds to the two domains unfolding at 341.6 +/- 0.6 K and one ligand binds to the domain unfolding at 347.8 +/- 0.6 K.