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PubMed1993

Angiotensin II receptor antagonists.

The development of novel non-peptide compounds with high affinity for-angiotensin II (Ang II) receptors has greatly facilitated the subclassification of Ang II receptors into AT1- and AT2-receptor subtypes. Whereas PD 123177 (1-(4-amino-3-methylphenyl)methyl-5-diphenyl-acetyl-4,5,6,7-tetrahydro- 1H-imidazol [4,5-c]pyridine-6-carboxylic acid) is the prototypical antagonist for AT2-receptors, DuP 753 (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1 H-tetrazol-5-yl)biphenyl-4-yl) methyl) imidazole, losartan) is the prototypical antagonist for AT1-receptors. So far, non-selective non-peptide Ang II receptor antagonists have not been identified although AT1/AT2 selectivity ratios of 17 and 37 have already been reported for BIBS 39 (4'-[(2-n-butyl-6- cyclohexylaminocarbonylamino-benzimidazole-1-yl)-methyl] biphenyl-2-carboxylic acid) and BIBS 222 (2-n-butyl-1-[4-(6-carboxy-2, 5-dichlorobenzoylamino)-benzyl]-6-N-(methylaminocarbonyl)- n-pentylaminobenzimidazole). Functional studies with AT1-antagonists indicate that Ang II antagonism at the receptor level can be rather complex. Experimental data suggests that not only are receptor binding kinetics involved, but also that additional binding sites, and possibly even AT1 subtypes, are involved. The antihypertensive activity of the AT1-antagonist DuP 753 is demonstrated in a high renin (2K 1C) and a low renin (TGRmREN2) hypertensive rat model. The kidney especially is very sensitive to Ang II and this organ seems to be a target for Ang II receptor antagonists. This can be demonstrated with experiments on the isolated rat kidney.

Amino Acid Sequence↗
PubMedDate not supplied

Pharmacological actions of essential (zinc, copper) and toxic metal ions (lead, mercury) on neurons and neuronal synapses of the snail, Helix pomatia L.

Actions of zinc, copper, lead and mercury ions on identified neurons and neuronal synapses of the snail, Helix pomatia L. were examined by use of electrophysiological methods. The studied metal ions depolarized the membrane and decreased the resistance but the neuronal excitability changed differentially. Copper and zinc transiently increased the amplitude and/or frequency of the spontaneous EPSPs or EPSCs in time- and dose-dependent ways. Lead attenuated the evoked EPSPs or EPSCs but copper and in some neurons zinc and mercury induced dose-dependent biphasic effects. Metal ions influenced the potential dependence, reversal potential, rate of rise and rate of fall of evoked responses differentially. The actions of zinc and lead on evoked EPSPs were reversible but those of copper and mercury were not and DTT treatment was ineffective. The affinity of the metal ions to the binding site(s) was low and the binding kinetics slow. The sensitivity of the binding site decreased in the order of mercury>copper>lead>zinc according to the IC50 values. The binding stoichiometry (nH value) changed from 1.4 to 2.6 for the various metal ions.

Animals↗
PubMed2005

Analysis of a family of antibodies with different half-lives in mice fails to find a correlation between affinity for FcRn and serum half-life.

In this study we analyzed mouse FcRn binding to different recombinant chimeric antibodies with human constant regions. This system has the advantage that in vivo half-life in animals expressing the receptor can be directly correlated with receptor binding kinetics. The goal was to determine which FcRn binding parameters, if any, correlate with the serum half-life of antibodies. We used a BIAcore surface plasmon resonance (SPR) device to study kinetic properties at different pHs and concentrations. The data were analyzed using a new model, the dual bivalent analyte model (DBVA), which postulates that there are two types of FcRn bound to the chip, one low affinity and one high affinity. In addition, it takes into consideration the possibility that the ligand, immunoglobulin G (IgG), can exist as both monomer and as higher molecular forms. While some antibodies bind to FcRn with different kinetics, including antibodies that differ only by containing the kappa or lambda light chain--a result which itself is unexpected--we cannot identify a single FcRn binding parameter that directly correlates with Ab half-life. Importantly, we demonstrate that some IgGs with higher affinity for FcRn do not have extended in vivo half-lives.

Animals↗
PubMed2005

Structure and kinetics of a transient antibody binding intermediate reveal a kinetic discrimination mechanism in antigen recognition.

Induced fit is a predominant phenomenon in protein-ligand interactions, yet it is invariably attributed without establishing the existence, let alone the structure, of the initial, low-affinity encounter complex. We determined the crystal structure of the encounter complex on the pathway of ligand binding by IgE antibody SPE7. We show that this complex is formed by a wide range of ligands that initially bind with identical affinity. Nonspecific ligands rapidly dissociate, whereupon the antibody isomerizes to a nonbinding isomer. Specific ligand complexes, however, slowly isomerize to give a high-affinity complex. This isomerization involves backbone and side-chain rearrangements of up to 14 A and the formation of specific hydrogen bonds. The postbinding conformational switch, combined with the prebinding isomerization to an energetically favorable nonbinding isomer, results in a "kinetic discrimination" mechanism that mediates selective binding, by a factor of >10(3), between highly related ligands that initially bind with the same affinity. This model may apply to proteins that bind multiple ligands in a specific manner or other proteins that, although capable of binding many ligands, are activated by only a few.

Anthracenes↗
PubMed2001

Analysing structure-function relationships with biosensors.

Elucidating the nature of the relationship between the structure and function of biomolecules remains one of the major challenges in biology. Biomolecules are dynamic entities that possess a variety of structures, and their functions at the molecular, cellular and organismic levels are quite different. Since there is no single causal link between structure and function, the search should be for correlations rather than causal relations. Biosensor instruments based on surface plasmon resonance are widely used for establishing correlations between the chemical structure of binding sites and their binding activity. Mutagenesis studies have shown that only a small percentage of the residues located in a binding site contribute to the binding energy. Since substitutions in residues located far away from the binding site are able to affect binding activity, this greatly complicates the rational design of proteins endowed with improved functions. However, biosensors can be used to determine and predict the influence of the chemical environment and of the structure of a ligand on binding kinetics.

Binding Sites↗
PubMed1990

Nonenzymatic glycosylation of HDL resulting in inhibition of high-affinity binding to cultured human fibroblasts.

Nonenzymatic glycosylation of plasma proteins may contribute to the excess risk of developing atherosclerosis in patients with diabetes mellitus. Because high-density lipoprotein (HDL) is believed to protect against atherosclerosis and is glycosylated at increased levels in diabetic individuals, the effects of nonenzymatic glycosylation of HDL3 on binding of HDL3 to cultured fibroblasts and to the candidate HDL-receptor protein were examined. HDL3 was glycosylated in vitro with glucose alone or in combination with sodium cyanoborohydride. With this catalyst, up to 40-50% of the lysine residues could be glycosylated, resulting in a progressive drop to nearly 60% in high-affinity binding to cultured fibroblasts at 4 degrees C. Binding to the 110,000-Mr candidate HDL-receptor protein was reduced by almost 75%. At levels of HDL glycosylation equivalent to the 3-5% observed in diabetes, high-affinity binding to fibroblasts at 4 degrees C was diminished by up to 15-20%. Binding kinetic studies paradoxically suggested that glycosylated HDL3 binds with higher affinity to a reduced number of binding sites. The findings in this study suggest that nonenzymatically glycosylated HDL may be functionally abnormal and might contribute to the development of atherosclerosis in patients with diabetes mellitus.

Animals↗
PubMed2004

Contribution of a conserved phenylalanine residue to the activity of Escherichia coli uracil DNA glycosylase.

Uracil DNA glycosylase (UDG) excises uracil from DNA to initiate repair of this lesion. This important DNA repair enzyme is conserved in viruses, bacteria, and eukaryotes. One residue that is conserved among all the members of the UDG family is a phenylalanine that stacks with uracil when it is flipped out of the DNA helix into the enzyme active site. To determine what contribution this conserved Phe residue makes to the activity of UDG, Phe-77 in the Escherichia coli enzyme was mutated to three different amino acid residues, alanine (UDG-F77A), asparagine (UDG-F77N), and tyrosine (UDG-F77Y). The effects of these mutations were measured on the steady-state and pre-steady-state kinetics of uracil excision in addition to enzyme.DNA binding kinetics. The overall excision activity of each of the mutants was reduced relative to the wild-type enzyme; however, each mutation gave rise to a different kinetic phenotype with different effects on substrate binding and catalysis. The excision activity of UDG-F77N was the most severely compromised, but this enzyme still bound to uracil-containing DNA at about the same rate as wild-type UDG. In contrast, the decrease in the excision activity of UDG-F77A is likely to reflect a greater reduction in uracil-DNA binding than in the catalytic step. Overall, the effects of the mutations on catalysis are best correlated with the polarity of the substituted residue such that an increase in polarity decreases the efficiency of uracil excision.

Catalysis↗
PubMed2005

A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex.

The structure of the scFv fragment FITC-E2, obtained from a naive phage antibody scFv library derived from human donors, was determined at 2.1 A resolution in the free form and at 3.0 A in the complexed form. The wild-type (wt) scFv binds fluorescein with a K(D) of 0.75 nM. The free scFv readily crystallizes by compacting its 18 amino acid-long CDR-H3, partially occluding the binding site and further blocking access by binding to the "bottom" of a neighboring scFv molecule with a cluster of exposed aromatic residues within CDR-H3. Only upon mutating one of the residues involved in this dominant crystal contact, an exposed tryptophan in the middle of CDR-H3, crystals of the complex could be obtained. A series of alanine mutants within the putative antigen binding site, covering a range of binding affinities, were used to relate macroscopic thermodynamic and kinetic binding parameters to single-molecule disruption forces measured by AFM. The effects of the mutations on the binding properties, particularly on the fraction of binding-competent molecules within the population, cannot be fully explained by changes in the strength of local interactions. The significant conformational change of CDR-H3 between the free and the liganded form illustrates the plasticity of the binding site. An accompanying study in this issue by Curcio and colleagues presents the molecular dynamics simulation of the forced unbinding experiments and explores possible effects of the mutations on the unbinding pathway of the hapten.

Antibodies, Monoclonal↗
PubMedDate not supplied

Kinetic evidence for isomerization of the dopamine receptor-raclopride complex.

The binding kinetics of the specific dopamine D2 antagonist [3H]raclopride to dopamine D2 receptors in rat neostriatum were studied. The pseudo-first-order rate constants of [3H]raclopride binding with these membranes revealed a hyperbolic dependence upon the antagonist concentration, indicating that the reaction had at least two consecutive and kinetically distinguishable steps. The first step was fast binding equilibrium, characterized by the dissociation constant KA = 12 +/- 3 nM. The following step corresponded to a slow isomerization of the receptor-antagonist complex, characterized by the isomerization equilibrium constant Ki = 0.11. The dissociation constant Kd = 1.3 nM, calculated from these kinetic data, was similar to Kd = 2.4 nM, determined from equilibrium binding isotherm for the radioligand. Implications of the complex reaction mechanism on dopamine D2 receptor assay by [3H]raclopride were discussed.

Animals↗
PubMed1985

Analysis of ligand binding to receptors by competition kinetics. Application to muscarinic antagonists in rat brain cortex.

We propose a method for obtaining kinetic parameters of unlabeled ligands which is based on analyzing the effect of their competition on the binding kinetics of a labeled ligand. In this method (competition kinetics), the binding kinetics of a labeled ligand are measured in the absence and in the presence of given concentrations of a competing unlabeled ligand. The rate equations appropriate to the particular kinetic model may be solved employing linear homogeneous differential equations. The rate constants appearing in the final integrated expressions can be evaluated by nonlinear regression. We have demonstrated the validity and applicability of the method to study receptor systems, using the muscarinic receptors in rat brain cortex as an experimental system. We have determined the various kinetic parameters of two muscarinic antagonists (N-methyl-4-[3H]piperidyl benzilate and (-)-N-[3H]methylscopolamine) by following directly their binding kinetics. These parameters were compared with those obtained for the same unlabeled ligands in homogeneous and heterogeneous competition studies. In all cases, the parameters obtained by the competition kinetics method were close to those extracted from direct binding kinetics, and in cases where both types of studies were performed on the same preparations, the parameters obtained by the two methods were essentially identical.

Animals↗
PubMed1997

Kinetics of competitive binding with application to thrombin complexes.

The kinetics of competitive binding is treated analytically, allowing the rate constants to be determined accurately from simple experiments. The method is especially suited to situations where traditional approximations and numerical integration fail, e.g., when the dissociation constants are small or when the concentration of one receptor cannot be measured accurately. The method is applied to the competitive binding of hirudin to thrombin and anhydrothrombin and found to be accurate to a few parts in ten million. The fitted rate constants show that anhydrothrombin binds hirudin more weakly than thrombin, with a 2.6-fold increase in its dissociation constant. The small relative difference in binding free energy (0.6 kcal/mol indicates that anhydrothrombin is structurally similar to thrombin.

Binding, Competitive↗
PubMed2005

Low concentrations of neuroactive steroids alter kinetics of [3H]ifenprodil binding to the NMDA receptor in rat frontal cortex.

The modulatory effects of the two neurosteroids pregnenolone sulphate (PS) and pregnanolone sulphate (3alpha5betaS) on [3H]ifenprodil binding to the N-methyl-D-aspartate (NMDA) receptor in rat frontal cortex were studied. The binding for [3H]ifenprodil itself displayed monophasic kinetics in all experiments. None of the neurosteroids displaced the radioligand from its binding site on the NR2B subunit of the NMDA receptor. However, their continual presence at nanomolar concentrations had significant effects on ligand binding kinetics, interacting through distinct sites in saturation, competition and dissociation experiments. PS at 30 nM enhanced the specific binding to about 150% of that in its absence and enhanced the dissociation rate three-fold indicating a positive modulation of [3H]ifenprodil binding to the NMDA receptor. Furthermore, PS increased Bmax and decreased Kd suggesting that the neurosteroid exposes new [3H]ifenprodil binding sites with altered properties. In contrast, 3alpha5betaS (30 nM) decreased specific [3H]ifenprodil binding to approximately 40% of that determined for the radioligand alone. The presence of 3alpha5betaS at nanomolar concentrations induced biphasic curve fits in saturation, competition as well as dissociation experiments. In conclusion, the present study show that the allosteric modulators PS or 3alpha5betaS change [3H]ifenprodil binding kinetics in a way indicating conformational alteration of its binding site on the NR2B subunit.

Animals↗
PubMed2005

Study of MMLV RT- binding with DNA using surface plasmon resonance biosensor.

Surface plasmon resonance biosensor technique was used to study the binding of Moloney murine leukemia virus reverse transcriptase without RNase H domain (MMLV RT-) with DNA in the absence and in the presence of inhibitors. Different DNA substrates, including single-stranded DNA (ssDNA), DNA template-primer (T-P) duplex and gapped DNA, were immobilized on the biosensor chip surface using streptavidin-biotin, and MMLV RT(-)-DNA binding kinetics were analyzed by different models. MMLV RT-; could bind with ssDNA and the binding was involved in conformation change. MMLV RT-; binding DNA T-P duplex and gapped DNA could be analyzed using the simple 1:1 Langmuir model. The lack of RNase H domain reduced the affinity between MMLV RT-; and T-P duplex. The effects of RT inhibitors, including efavirenz, nevirapine and quercetin, on the interaction between MMLV RT-; and gapped DNA were analyzed according to recovered kinetics parameters. Efavirenz slightly interfered with the binding between RT and DNA and the affinity constant in the presence of the inhibitor (K(A) = 1.21 x 10(6) M(-1)) was lower than in the absence of the inhibitor (KA = 4.61 x 10(6) M(-1)). Nevirapine induced relatively tight binding between RT and DNA and the affinity constant in the presence of the inhibitor (K(A) = 1.47 x 10(7) M(-1)) was approximately three folds higher than without nevirapine, mainly due to rapid association and slow dissociation. Quercetin, a flavonoid originating from plant which has previously shown strong inhibition of the activity of RT, was found to have minimal effect on the RT-DNA binding.

Alkynes↗
PubMed1989

[125]I-spectramide: a novel benzamide displaying potent and selective effects at the D2 dopamine receptor.

The new substituted benzamide Spectramide, (N-[2-[4-iodobenzyl-N-methylamino]-2-methoxy-4-ethyl]-5-chloro- methylamine] benzamide) labelled with 125I was used as a potent and highly selective dopamine-D2 receptor antagonist in rat striatal homogenates for in vitro receptor binding. Kinetic experiments demonstrated the reversibility of the binding and the estimated Kd from saturation analysis was 25 pM, with a Bmax of 20 pmol/g of tissue. Competition studies showed that spectramide did not interact potently with the D1 or dopamine-uptake site. Drugs known to interact with other receptor systems were weak competitors of the binding, while binding was potently inhibited by other D2 antagonists, such as spiperone and eticlopride. These data indicate that Spectramide binds selectively and with high affinity to the dopamine D2 receptors, and may prove to be a useful tool for the study of these receptors in vivo using PET or SPECT.

Animals↗
PubMed1996

Tryptophan 60-D in the B-insertion loop of thrombin modulates the thrombin-antithrombin reaction.

In a recent study it was demonstrated that thrombin des-PPW reacts with antithrombin (AT) very poorly. In this study it is shown that a Trp to Ala (W60A) mutant of thrombin also reacts with AT at a lower rate than thrombin. The inhibition kinetics were studied by the slow-binding kinetic approach. In both the presence and absence of heparin, the pseudo-first-order rate constant of thrombin inhibition (kobs) increased linearly with AT concentration, indicating that inhibition, in the concentration range covered, conforms to a bimolecular reaction E+I-->K assn E-I. Only the second-order association rate constant (kassn) for thrombin can be estimated [6.8 +/- 2.7) x 10(3) M-1 s-1 in the absence of heparin and (4.1 +/- 1.2) x 10(6) M-1 s-1 in the presence of heparin]. With W60A and des-PPW, the kobs of inhibition increased hyperbolically as a function of AT concentration, indicating that the inhibition is a two-step process according to E+I<==>K init E.I-->k2 E-I. The kinetic constants for W60A were estimated to be Kinit = 13.6 +/- 3.3 microM and k2 = 0.007 +/- 0.001 s-1 in the absence of heparin and K init = 13.6 +/- 3.1 nM and k2 = 0.008 +/- 0.002 s-1 in the presence of heparin. AT inhibited des-PPW very slowly [K assn = (2.9 +/- 0.7) x 10(1) M-1 s-1], but heparin accelerated the reaction approximately 20,000-fold and made it possible to demonstrate a two-step reaction mechanism for des-PPW with K init = 10.4 +/- 2.3 nM and k2 = 0.006 +/- 0.001 s-1. In contrast to thrombin, an active AT-binding pentasaccharide enhanced the inhibition of des-PPW approximately 15-fold. These results indicate that (1) in contrast to thrombin, the heparin-induced conformational change in AT is required for optimal inhibition of des-PPW and (1) Trp60 is essential for normal thrombin-AT reaction. On the basis of these results, a modified model for thrombin-AT interaction is proposed.

Animals↗
PubMed2000

Kinetic determination of focal adhesion protein formation.

I examined the binding kinetics between integrin (alpha(IIb)beta(3)) and purified focal adhesion proteins, including alpha-actinin, filamin, vinculin, talin, and F-actin. Using static light-scatter technique, I observed affinities of the order talin > filamin > F-actin > alpha-actinin > (talin when bound to vinculin) which were lower when integrin was complexed with fibronectin. No binding between integrin and vinculin was detected. The calculated dissociation constants (K(d)) ranged between 0.4 microM and 5 microM. These results in part confirm previously published data using different methods. The modest affinity with which the focal adhesion proteins interact in vitro might be indicative of how cells, e.g., thrombocytes, gain a high degree of versatility and velocity.

Actinin↗
PubMed1992

The kinetics of mebendazole binding to Haemonchus contortus tubulin.

The kinetics of the binding of mebendazole (MBZ) to tubulin from the third-stage (L3) larvae of the parasitic nematode, Haemonchus contortus, have been characterized. In partially purified preparations, the association of [3H]MBZ to nematode tubulin was rapid, k1 = (2.6 +/- 0.3) x 10(5) M-1 min-1, but dissociation was slow, k-1 = (1.58 +/- 0.02) x 10(-3) min-1. The affinity constant (K(a)) for the interaction, determined by the ratio k1/k-1, was (1.6 +/- 0.2) x 10(8) M-1. Similar results were obtained with crude cytosolic fractions. In equilibrium studies, performed with partially purified nematode tubulin under similar conditions, a K(a) of (5.3 +/- 1.6) x 10(6) M-1 was obtained. The best estimate for the K(a) of the MBZ-nematode tubulin interaction is considered to be the 'kinetic' value determined from the ratio of rate constants. The slow dissociation of MBZ from nematode tubulin, which contrasts with the rapid dissociation of MBZ from mammalian tubulin, supports the hypothesis that the selective toxicity of the benzimidazole anthelmintics results from a difference between the affinities of mammalian and nematode tubulins for these drugs.

Animals↗
PubMed1986

The structural bases for the unique ligand binding properties of Glycera dibranchiata hemoglobins. A resonance Raman study.

The hemoglobin of the marine annelid Glycera dibranchiata possesses several unique features: the hemoglobin consists of multiple monomeric and polymeric components, quaternary structure is lacking, the distal histidine is replaced by leucine in at least one monomeric constituent, and 4) the protein exhibits extremely rapid ligand binding kinetics. The effect of these structural modifications on the ligand binding process has been evaluated using resonance Raman spectroscopy to examine the vibrational modes of the porphyrin macrocycle in deoxy and carbonmonoxy equilibrium species of hemoglobin G. dibranchiata in both the unseparated monomeric and polymeric forms and in a single monomeric component designated Fraction II. Significant differences relative to hemoglobin were found in porphyrin pi electron density, vinyl environment, low frequency vibrational modes, and, in particular, the Fe-proximal histidine stretching mode. Spectra of the deoxy heme transients generated within 10 ns of ligand photolysis have also been examined. These clearly indicate large differences in the heme pocket dynamics subsequent to CO photolysis in G. dibranchiata hemoglobins relative to other hemoglobins. The significance of these results in terms of the kinetics and thermodynamics of ligand binding is discussed.

Animals↗