Ligand Saturation Research Articles

Evidence that annexin II is not a putative membrane receptor for 1alpha,25(OH)2-vitamin D3.

The seco-steroid hormone 1alpha,25(OH)(2)-vitamin D(3) (1,25-D(3)) is known to generate biological responses via both genomic and non-genomic rapid signal transduction pathways. The calcium regulated annexin II/p11 heterotetramer (AII(2)/p11(2)] was proposed by Baran and co-authors to be the membrane receptor responsible for mediating non-genomic, rapid actions of 1,25-D(3), based on ligand affinity labeling, competition, and saturation analysis experiments. Given the cytosolic presence of both the monomeric and heterotetrameric form of AII and their functional regulation by intracellular calcium concentrations, which are known to be affected by 1,25-D(3) rapid, non-genomic activities, we investigated in vitro the affinity of [(3)H]1,25-D(3) for the AII monomer and AII(2)/p11(2) in the absence and presence of calcium using saturation analysis and gel-filtration chromatography. Using two different techniques for separating bound from free ligand (perchlorate and hydroxylapatite (HAP)) over a series of 30 experiments, no evidence for specific binding of [(3)H]1,25-D(3) was obtained with or without the presence of 700 nM exogenous calcium, using either the AII monomer or AII(2)/p11(2). However saturable binding of [(3)H]1,25-D(3) to the lipid raft/caveolae enriched rat intestinal fraction was consistently observed (K(d) = 3.0 nM; B(max) = 45 fmols/mg total protein). AII was detected in lipid raft/caveolae enriched fractions from rat and mouse intestine and ROS 17/2.8 and NB4 cells by Western blot, but incubation in the presence of exogenous calcium did not ablate 1,25-D(3) binding as reported by Baran et al. Our results suggest that AII does not bind 1,25-D(3) in a physiologically relevant manner; however, recent studies linking AII(2)/p11(2) phosphorylation to vesicle fusion and its calcium regulated localization may make AII a possible down-stream substrate for 1,25-D(3) induced rapid cellular effects.

Syntheses, Properties, and X-ray Crystal Structures of Piano-Stool Iron Complexes Bearing an N-Heterocyclic Carbene Ligand

Piano-stool iron complexes containing the C−C unsaturated IMes or saturated H2-IMes ligand are readily synthesized from [CpFe(CO)2(I)]. Substitution of the iodide ligand affords the cationic specie...

Improved palladium-catalyzed coupling reactions of aryl halides using saturated N-heterocarbene ligands

Abstract The incorporation of saturated N -heterocyclic carbenes into palladium pre-catalysts gives high catalyst activity in the Suzuki coupling of aryl iodides, bromides and deactivated aryl chloride substrates, whereas the yield of the palladium catalyzed Heck reaction of deactivated aryl chlorides is negligible. The complexes were generated in the presence of Pd(OAc) 2 by in situ deprotonation of 1,3-dialkylimidazolinium salts LHX ( 1 ) which were characterized by conventional spectroscopic methods and elemental analyses.

A Combined Experimental and Theoretical Study Examining the Binding of N-Heterocyclic Carbenes (NHC) to the Cp*RuCl (Cp* = η5-C5Me5) Moiety: Insight into Stereoelectronic Differences between Unsaturated and Saturated NHC Ligands

Combined solution calorimetric and quantum mechanics studies of reactions involving saturated and unsaturated N-heterocyclic carbene (NHC) ligands show that the difference in their relative bond dissociation energies is very small (1 kcal·mol-1). Structural and computational studies reveal small metric parameter differences. These observations in conjunction with relative reactivity profiles of NHC-modified ruthenium-based olefin metathesis catalysts suggest that very small changes in the donor properties of the NHC ligands can translate into significant differences in catalytic properties.

Expression of a soluble glycine binding domain of the NMDA receptor in Escherichia coli

Glycine is an essential co-agonist of the excitatory N-methyl- d-aspartate (NMDA) receptor. The glycine binding site of this subtype of ionotropic glutamate receptors is formed by the S1 and S2 regions of the NR1 subunit. Here, different S1S2 fusion proteins were expressed and purified from Escherichia coli cultures, and refolding protocols were established allowing the production of 30 mg of soluble S1S2 fusion protein from 1 liter bacterial culture. After affinity purification and renaturation, two of the fusion proteins (S1S2 and S1S2-V1) bound the competitive glycine site antagonist [ 3H]MDL105,519 with K d values of 9.35 and 3.9 nM, respectively. In contrast, with three other constructs (S1S2M, S1S2-V2, and -V3) saturable ligand binding could not be obtained. These results redefine the S1S2 domains required for high-affinity glycine binding. Furthermore, our high-affinity binding proteins may be used for the large-scale production of the glycine binding core region for future structural studies.

Expression and molecular pharmacology of the mouse CRTH2 receptor.

Prostaglandin D2 (PGD2), the predominant prostanoid produced by activated mast cells, is implicated in a variety of allergic diseases. PGD2 exerts its effects through two G-protein coupled receptors, DP and CRTH2. PGD2 mediates chemotaxis of eosinophils, basophils, and Th2 cells via CRTH2-evoked signaling, suggesting a role for this receptor in allergic disease. To characterize the mouse CRTH2 ortholog (mCRTH2), we amplified the mCRTH2 receptor gene and expressed it in HEK293 cells. Saturation ligand binding isotherms demonstrated high-affinity binding of [3H]PGD2, with a Kd of 8.8 +/- 0.8 nM. Competition binding assays with a panel unlabeled prostanoids demonstrated an order of affinity of 13,14-dihydro-15-keto-PGD2 (DK-PGD2) >or= 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) >or= PGD2 >or= PGJ2. [3H]PGD2 binding was also displaced by the nonsteroidal anti-inflammatory drug indomethacin, with a Ki value of 1.04 +/- 0.13 microM. No [3H]PGD2 displacement was detected using fluribrofen, ibuprofen, or aspirin as competitors at concentrations of up to 30 microM. PGD2, DK-PGD2, 15d-PGJ2, and indomethacin each inhibited intracellular cAMP generation in stable transfectant ER293/mCRTH2 cells through a pertussis toxin (PTX) sensitive pathway, consistent with mCRTH2 coupling to a Gi heterotrimeric G-protein. Activation of mCRTH2 elicited chemotaxis of ER293/mCRTH2 cells in response to PGD2, indomethacin, and 15d-PGJ2. mCRTH2-dependent chemotaxis was inhibited by PTX and wortmannin, indicating dependence on Gi and PI 3-kinase signal transduction pathways. These data provide the first pharmacological and functional characterization of the mouse CRTH2 receptor.

Specificity in template syntheses of hexaaza-macrobicyclic cages: [Pt(Me5-tricosatrieneN6)]4+ and [Pt(Me5-tricosaneN6)]4+.

The racemic C3 hexadentate cage complex, [Pt(Me5-tricosatrieneN6)]Cl4 (1,5,9,13,20-pentamethyl-3,7,11,15,18,22-hexaazabicyclo[7.7.7]tricosa- 3,14,18-triene)platinum(IV) tetrachloride), was synthesised stereospecifically and regiospecifically from a reaction of the bis-triamine template [Pt(tamc)2]Cl4 (bis[1,1,1-tris(aminomethyl)ethane]- platinum(IV) tetrachloride) with formaldehyde and then propanal, in acetonitrile under basic conditions. Largely, one racemic diastereoisomer was obtained in a surprisingly high yield (approximately 50%), even though the molecule has seven chiral centres. The origins of the stereoselective synthesis are addressed. The crystal structure of [Pt(Me5-tricosatrieneN6)]-(ZnCl4)1.5Cl.2H2O showed that all three imines were attached to one tame fragment with a chiral amine site ([symbol: see text] SSS, delta RRR) and a chiral methine carbon site ([symbol: see text] RRR, delta SSS) on each ligand strand. The PtN6(4+) moiety had a slightly distorted octahedral configuration with the two types of Pt-N bonds related to the imine and the amine donors, 2.050(7) and 2.072(6) A, respectively. Treatment with sodium borohydride (15 s, 20 degrees C) at pH approximately 12.5 reduced the imine groups, but not the Pt(IV) ion, producing a C3 saturated ligand complex [Pt(Me5-tricosaneN6)]Cl4 ((1,5,9,13,20- pentamethyl-3,7,11,15,18,22- hexaazabicyclo[7.7.7]tricosane)platinum(IV)tetrachloride). X-ray crystallographic analysis showed that the average Pt-N bond distance in the cation increased upon imine reduction to 2.10 (av) A. The cyclic voltammograms of the two cage complexes displayed irreversible two-electron reduction waves in aqueous media and a approximately 0.3 V shift to more positive potentials compared to that of the smaller cavity sar (3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane) analogue. After reduction, net dissociation of one strand of the cage was also evident, to give unstable square planar Pt(II) macrocyclic products.

Pharmacological investigation of the role of ion channels in salivary secretion.

The role of K+ and Cl- channels in salivary secretion was investigated, with emphasis on the potential role of Ca2+ -activated K+ channels. Ligand saturation kinetic assays and autoradiography showed large-conductance (BK) K+ channels to be highly expressed in rat submandibular and parotid glands, whereas low-conductance (SK) K+ channels could not be detected. To investigate the role of K+ and Cl- channels in secretion, intact rabbit submandibular glands were vascularly perfused and secretion induced by 10 microM ACh. Secretion was inhibited by 34+/-3% following perfusion with the general K+ channel inhibitor Ba2+ (5 mM), whereas organic inhibitors of BK (200 nM paxilline) or intermediate-conductance (IK) K+ channels (5 microM clotrimazole) had no effect. Secretion was strongly influenced by Cl- channel inhibitors, as 100 microM 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) completely abolished, while 10 microM NPPB, 20 microM NS1652 and 20 microM NS3623 reduced secretion by 34+/-3%, 23+/-3% and 59+/-4%, respectively. In conclusion, although high expression levels of BK channels were demonstrated, pharmacological tools failed to demonstrate any role for BK, IK or SK channels in salivary secretion in the rabbit submandibular gland. Other types of K+ channel, however, and particularly Cl- channels, are essential for ACh-induced salivary secretion.

Indirect capillary electrophoresis with 8-anilino-1-naphthalenesulfonic acid as a fluorescence probe for determining the apparent stability constant of an inclusion complex formed between a cyclodextrin and a solute

An indirect capillary electrophoresis (CE) method was developed based on two competitive chemical equilibria for determining the stability constant of an inclusion complex formed between a cyclodextrin and a solute. 8-Anilino-1-naphthalenesulfonic acid was employed as a fluorescence probe. A linear relationship between mobility difference and concentration of uncomplexed ligand was theoretically established and experimentally verified. The principle of the method was explained using an example of determining stability constant of an inclusion complex formed between a ligand of hydroxypropyl-β-cyclodextrin and a solute of amantadine. The stability constant was determined to be ∼2·10 2 M −1. It was calculated without knowledge of the mobility of the complex measured at saturating ligand concentrations. This indirect method can be applied to solutes and ligands lacking signal response on the selected detector in the CE. In addition, the indirect method is valid for both charged and neutral solutes and ligands.

Intervalence charge transfer transition in mixed valence complexes synthesised from RuIII(edta)- and FeII(CN)5-cores

Intervalence charge transfer properties were studied for a set of mixed valence complexes incorporating Ru(III) and Fe(II)-centres linked by various saturated and unsaturated bridging ligands (BL). Studies reveal that degree of ground state electronic interaction and coupling between Ru(III) and Fe(II)-centrescanbe attenuated by changing the nature of the bridging ligand. Further, inclusion of the bridging ligand with interrupted π-electron system in a β-CD cavity initiate an optical electron transfer from Fe(II) to Ru(III) which is otherwise not observed.

Cobalt(III) Acetylacetonate Chemisorbed on Aluminum-Nitride-Modified Silica: Characteristics and Hydroformylation Activity

Co/AlN/SiO2 catalysts were prepared by the saturative chemisorption of cobalt(III) acetylacetonate (Co(acac)3). The support was bare silica or silica that had been modified with aluminum nitride (AlN) by repeated separate, saturated chemisorptions of trimethylaluminum and ammonia two or six times. Chemisorption of Co(acac)3 occurred on all the supports up to a saturation ligand density of 2.7 acac nm-2; the amount of bonded cobalt decreased from 2.1 to 1.5 atCo nm-2 with increasing extent of AlN modification of the support. Ligand exchange reaction, releasing Hacac, occurred less on AlN-modified silica than on bare silica. This induced difference in the reduction behavior of the catalysts, and catalytic activity in gas-phase hydroformylation of ethene, was lower with Co/AlN/SiO2 than with Co/SiO2 catalyst.

A comparative study of olefin or acetylene insertion into Ru–H or Os–H of MHCl(CO)(phosphine)2

The adduct OsHCl(C2D4)(CO)L2 (L = PiPr3) shows only very slow H/D exchange at 25 °C, but this is easily detectable at 65 °C; no ethyl species is formed in detectable concentration. RuHCl(CO)L2 shows no detectable C2D4 adduct, but Ru–H/C–D exchange at 60 °C is actually faster than for Os. DFT (B3PW91) calculations have been carried out to analyze the relative energies of the isomeric forms that would result from the addition of an alkene or an alkyne to MH(Cl)(CO)(PH3)2 (M = Os, Ru). Thus, 18-electron alkyne adducts are compared to the 18-electron vinylidene isomer and to the 16-electron vinyl complex. Similarly, the 18-electron alkene adduct is compared to the 18-electron carbene isomer and to the 16-electron ethyl complex. Two factors are found to control the products formed: (i) the Os complex favors unsaturated π-bonded ligands and an 18-electron count while Ru favors saturated ligands and an unsaturated metal center; (ii) the weaker π bond in the alkyne than in the alkene makes insertion or isomerization of an alkyne thermodynamically more favored than that of an alkene. This results in ethyl complexes being less favored than vinyl complexes in similar experimental conditions. For RuHCl(CO)L2′, where L′ is PiPr2[3,5-(CF3)2C6H3], 1 atm ethylene gives a detectable, colorless ethylene adduct, then also a detectable ethyl complex, all in facile equilibrium.

Linking ecological impact to metal concentrations and speciation: A microcosm experiment using a salt marsh meiofaunal community

Microcosm experiments addressed the impact of a mixture of Cu, Cr, Cd, Pb, and Hg at three concentrations after 36 h, 12 d, and 30 d on a meiofauna-dominated salt marsh community. In addition to analyzing effects on meiofaunal abundances, the study quantified the sediment metal concentrations of all five metals and pore-water concentrations, speciation, and ligand complexation of Cu. Abundances of deposit feeders such as the polychaete Streblospio benedicti, gastropods, and bivalves were impacted at lower metal concentrations than the mainly algal-feeding copepods, ostracods, and nematodes. We suggest that this might be due to bulk ingestion of metal-contaminated sediments resulting in relatively higher metal exposure in the deposit feeders than in the other, nondeposit feeding taxa. Copepod and ostracod abundances decreased only in the highest metal treatment, where levels of inorganic Cu ([Cu']) in pore waters were similar to levels associated with both acute and subacute toxicity in published in vivo toxicity studies of marine copepods. The higher metal treatments yielded disproportionately higher pore-water [Cu] compared with sediment [Cu], suggesting saturation of sediment-associated ligands with increased additions of Cu. Similarly, the higher metal treatments appeared to reach saturation of the organic Cu ligands, with the excess pore-water [Cu] present in the more toxic, inorganic species of Cu. Acid-volatile sulfide (AVS) concentrations at sediment horizons inhabited by meiofauna were low and AVS was not considered a significant metal ligand at these depths. Since meiofauna are predominantly associated with oxic surface sediments, it is doubtful that AVS is a major factor controlling availability of free metal for exposure to these taxa.

Interaction of beta-lactoglobulin with small hydrophobic ligands as monitored by fluorometry and equilibrium dialysis: nonlinear quenching effects related to protein--protein association.

Although a thorough characterization of binding parameters is essential for application of beta-lactoglobulin as a carrier for a variety of small hydrophobic ligands, the binding parameters derived in various studies using various techniques are inconsistent. The bindings of several small ligands as detected by fluorometry and equilibrium dialysis were compared. Fluorescence spectroscopy showed that beta-ionone, retinol, and fatty acid lactones all bound in the vicinity of a tryptophan residue. Retinol and fatty acid lactone competed for the same binding site. Exclusively for ligands that quench the beta-lactoglobulin fluorescence through a resonance energy transfer mechanism, fluorometry yielded a systematically higher binding affinity than equilibrium dialysis. The binding overestimation in fluorometric measurements can be explained by oligomer formation of protein, together with an underestimation of the limiting quenching level at saturating ligand concentrations due to the use of a limited set of data points.

β-Cyclodextrin-Assisted Intervalence Charge Transfer in Mixed-Valent [2]Rotaxane Complexes Having Metal Centers Linked by an Interrupted π-Electron System.

FeII to RuIII electron transfer is initiated by the inclusion of saturated bridging ligands into the β-cyclodextrin (CD) cavity of [2]rotaxane complexes and is observed as an intervalence charge transfer (IVCT) band in the electronic spectra. The rotaxanes are synthesized by using RuIII and FeII complexes as stoppers, β-CD as the cyclic molecular head, and 4,4'-bipyridine derivatives with interrupted π-electron system as the bridging ligand between the metal centers.

Tmtacn, tacn, and triammine complexes of (eta 6-arene)OsII: syntheses, characterizations, and photosubstitution reactions (tmtacn = 1,4,7-trimethyl-1,4,7-triazacyclononane; tacn = 1,4,7-triazacyclononane).

A series of (eta 6-arene)OsII complexes containing the saturated nitrogen donor ligands tmtacn, tacn, and NH3 are prepared and characterized. The electrochemical properties and photochemical reactions of these complexes are studied, and the solid-state structures for [(eta 6-p-cymene)Os(tacn)](PF6)2 (1) and [(eta 6-p-cymene)Os(tmtacn)](PF6)2 (2) are determined. Single-crystal X-ray data: 1, orthorhombic, space group Pbca-D2h15 (No. 61), with a = 14.716(3) A, b = 17.844(3) A, c = 18.350(4) A, V = 4819(2) A3, and Z = 8; 2, monoclinic, space group C2-C2(3) (No. 5), with a = 17.322(4) A, b = 10.481(3) A, c = 15.049(4) A, beta = 98.72 degrees, V = 2701(1) A3, and Z = 4.

Photooxidation of nickel(II) macrocyclic complexes by mono- and biphotonic processes from the charge-transfer to solvent excited states in aqueous solutions

Excitation of nickel(II) macrocyclic complexes using 248 nm laser pulses leads to the formation of solvated electron in aqueous solutions. Formation of solvated electrons is confirmed by a characteristic absorption spectrum which shows a maximum at 680 nm. From the decay kinetics, bimolecular rate constants were determined for the decay of the solvated electron. Nickel(II) complexes with saturated macrocyclic ligands produce photoelectrons by biphotonic processes and the nickel(II) complex with unsaturated ligand ejects electrons from excited states by a monophotonic process as determined by varying the excitation energy of the laser.

120- and 160-kDa Receptors for Endogenous Mitogenic Peptide, Phytosulfokine-α, in Rice Plasma Membranes

Plant cells in culture secrete a sulfated peptide named phytosulfokine-alpha (PSK-alpha), and this peptide induces the cell division and/or cell differentiation by means of specific high and low affinity receptors. Putative receptor proteins for this autocrine type growth factor were identified by photoaffinity labeling of plasma membrane fractions derived from rice suspension cells. Incubation of membranes with a photoactivable (125)I-labeled PSK-alpha analog, [N(epsilon)-(4-azidosalicyl)Lys(5)]PSK-alpha (AS-PSK-alpha), followed by UV irradiation resulted in specific labeling of 120- and 160-kDa bands in SDS-polyacrylamide gel electrophoresis. The labeling of both bands was completely inhibited by unlabeled PSK-alpha and partially decreased by PSK-alpha analogs possessing moderate binding activities. In contrast, PSK-alpha analogs that have no biological activity showed no competition for (125)I-AS-PSK-alpha binding, confirming the specificity of binding proteins. Analysis of the affinity of (125)I incorporation into the protein by ligand saturation experiments gave apparent K(d) values of 5.0 nm for the 120-kDa band and 5.4 nm for the 160-kDa band, suggesting that both proteins correspond to the high affinity binding site. Treatment of (125)I-AS-PSK-alpha cross-linked proteins with peptide N-glycosidase F demonstrated that both proteins contained approximately 10 kDa of N-linked oligosaccharides. Specific cross-linking of (125)I-AS-PSK-alpha was also observed by using plasma membranes derived from carrot and tobacco cells, indicating the widespread occurrence of the binding proteins. Together, these data suggest that the 120- and 160-kDa proteins are PSK-alpha receptors that mediate the biological activities of PSK-alpha.

Partial IGF affinity of circulating N- and C-terminal fragments of human insulin-like growth factor binding protein-4 (IGFBP-4) and the disulfide bonding pattern of the C-terminal IGFBP-4 domain.

Within the IGF axis, the insulin-like growth factor-binding proteins (IGFBPs) are known to play a pivotal role in cell proliferation and differentiation. Defined proteolysis of the IGFBPs is proposed to be an essential mechanism for regulating IGF bioavailability. The generated IGFBP fragments in part exhibit different IGF-dependent and -independent biological activities. Characterizing naturally occurring forms of IGFBPs in human plasma, we identified both a N- and a C-terminal fragment of IGFBP-4 by means of immunoreactivity screening. As a source for peptide isolation, we used large amounts of human hemofiltrate obtained from patients with chronic renal failure. Purification of the IGFBP-4 peptides from hemofiltrate was performed by consecutive cation-exchange and reverse-phase chromatographic steps. Mass spectrometric and sequence analysis revealed an M(r) of 13 233 for the purified N-terminal fragment spanning residues Asp(1)-Phe(122) of IGFBP-4 and an M(r) of 11 344 for the C-terminal fragment extending from Lys(136) to Glu(237). Proteolytic digestion and subsequent biochemical analysis showed that the six cysteines of the C-terminal IGFBP-4 fragment are linked between residues 153-183, 194-205, and 207-228 (disulfide bonding pattern, 1-2, 3-4, and 5-6). Plasmon resonance spectroscopy, ligand blot analysis, and saturation and displacement studies demonstrated a very low affinity of the C-terminal IGFBP-4 fragment for the IGFs (IGF-II, K(d) = 690 nM; IGF-I, K(d) > 60 nM), whereas the N-terminal fragment retained significant IGF binding properties (IGF-II, K(d) = 17 nM; IGF-I, K(d) = 5 nM). This study provides the first molecular characterization of circulating human IGFBP-4 fragments formed in vivo exhibiting an at least 5-fold decrease in the affinity of the N-terminal IGFBP-4 fragment for the IGFs and a very low IGF binding capacity of the C-terminal fragment.

THE USE OF CELLULOSE BINDING DOMAIN-PROTEIN A FOR IMMUNOADSORPTION OF IgG

Immunoaffinity adsorption is increasingly used for protein purification and extracorporeal therapeutic applications. Immunoadsorption of IgG is under study for treatment of patients with high panel reactivity prior to transplantation. Synthetic membranes have advantages as support matrices compared to conventional gel media because they are incompressible, eliminate internal diffusion limitations, and permit plasma separation and immunoadsorption in the same device. The goal of this study was to explore the performance of the conjugate cellulose binding domain (CBD)-protein A bound non covalently to cellulose triacetate membranes for adsorption of IgG and to compare it with protein A attached covalently to polysulfone and nylon membranes. The binding constant for CBD-protein A interaction with IgG was measured with both static and dynamic methods. Breakthrough curves up to ligand saturation were measured to study the effect of IgG concentration, filtrate flow rate, and presence of albumin. Experiments with whole blood showed in-vitro feasibility of the concept. Binding capacity increased from 50 to the extraordinarily high value of 400 mg/ml membrane volume with CBD-protein A bound to cellulose triacetate fibers. The capacity was greater by a factor of 15 when compared to protein A attached to polysulfone and nylon membranes.