Apparent Dissociation Rate Constants Research Articles

Recognition of Phospholipids in Streptomyces Phospholipase D

To investigate the contribution of amino acid residues to the enzyme reaction of Streptomyces phospholipase D (PLD), we constructed a chimeric gene library between two highly homologous plds, which indicated different activity in transphosphatidylation, using RIBS (repeat-length independent and broad spectrum) in vivo DNA shuffling. By comparing the activities of chimeras, six candidate residues related to transphosphatidylation activity were shown. Based on the above result, we constructed several mutants to identify the key residues involved in the recognition of phospholipids. By kinetic analysis, we identified that Gly188 and Asp191 of PLD from Streptomyces septatus TH-2, which are not present in the highly conserved catalytic HXKXXXXD (HKD) motifs, are key amino acid residues related to the transphosphatidylation activity. To investigate the role of two residues in the recognition of phospholipids, the effects of these residues on binding to substrates were analyzed by surface plasmon spectroscopy. The result suggests that Gly188 and Asp191 are involved in the recognition of phospholipids in correlation with the N-terminal HKD motif. Furthermore, this study also provides experimental evidence that the N-terminal HKD motif contains the catalytic nucleophile, which attacks the phosphatidyl group of the substrate.

Design of a Novel Class of Peptide Inhibitors of Cyclin-dependent Kinase/Cyclin Activation

Cyclin dependent kinases (CDKs) are key regulators of the cell cycle progression and therefore constitute excellent targets for the design of anticancer agents. Most of the inhibitors identified to date inhibit kinase activity by interfering with the ATP-binding site of CDKs. We recently proposed that the protein/protein interface and conformational changes required in the molecular mechanism of CDK2-cyclin A activation were potential targets for the design of specific inhibitors of cell cycle progression. To this aim, we have designed and characterized a small peptide, termed C4, derived from amino acids 285-306 in the alpha5 helix of cyclin A. We demonstrate that this peptide does not interfere with complex formation but forms stable complexes with CDK2-cyclin A. The C4 peptide significantly inhibits kinase activity of complexes harboring CDK2 in a competitive fashion with respect to substrates but does not behave as an ATP antagonist. Moreover, when coupled with the protein transduction domain of Tat, the C4 peptide blocks the proliferation of tumor cell lines, thereby constituting a potent lead for the development of specific CDK-cyclin inhibitors.

Structural Basis of Citrate-dependent and Heparan Sulfate-mediated Cell Surface Retention of Cobra Cardiotoxin A3

Anionic citrate is a major component of venom, but the role of venom citrate in toxicity other than its inhibitory effect on the cation-dependent action of venom toxins is poorly understood. By immobilizing Chinese hamster ovary cells in microcapillary tubes and heparin on sensor chips, we demonstrated that heparan sulfate-mediated cell retention of the major cardiotoxin (CTX) from the Taiwan cobra, CTX A3, near membrane surfaces is citrate-dependent. X-ray determination of a CTX A3-heparin hexasaccharide complex structure at 2.4 A resolution revealed a molecular mechanism for toxin retention in which heparin-induced conformational changes of CTX A3 lead to citrate-mediated dimerization. A citrate ion bound to Lys-23 and Lys-31 near the tip of loop II stabilizes hydrophobic contact of the CTX A3 homodimer at the functionally important loop I and II regions. Additionally, the heparin hexasaccharide interacts with five CTX A3 molecules in the crystal structure, providing another mechanism whereby the toxin establishes a complex network of interactions that result in a strong interaction with cell surfaces presenting heparan sulfate. Our results suggest a novel role for venom citrate in biological activity and reveal a structural model that explains cell retention of cobra CTX A3 through heparan sulfate-CTX interactions.

In vitro characterization of novel NR2B selective NMDA receptor antagonists

N-Methyl- d-aspartate (NMDA) subunit specific receptor antagonism has potential therapeutic application for multiple CNS pathologies. MERCK 1, MERCK 2, and MERCK 3 are novel NR2B subtype selective NMDA receptor antagonists. The affinity and the kinetic mechanism of inhibition by these antagonists and ifenprodil were investigated using the whole-cell configuration of the patch clamp technique, calcium flux, and radioligand binding on a mouse cell line L(tk-) expressing recombinant human heteromeric NMDA receptors consisting of NR1a/NR2B subunit combinations. The rank order of potency, as determined by electrophysiology, was ifenprodil < MERCK 2 < MERCK 1 < MERCK 3 with K D's 79 ± 8, 2.4 ± 1.1, 1.3 ± 0.9, and ∼0.16 ± 0.02 nM, respectively. The apparent dissociation rate constants among these compounds differed by as much as 394-fold whereas the apparent association constants varied less than 3-fold. Higher affinities were a result of slower drug dissociation kinetics of receptor unbinding. Maximal inhibition was not voltage-dependent and was not statistically different at saturating concentrations by these compounds. These results provide the first detailed functional analysis of the kinetic mechanism of MERCK 1, MERCK 2, and MERCK 3 inhibition of NMDA receptors.

Diltiazem inhibits hKv1.5 and Kv4.3 currents at therapeutic concentrations

In the present study we examined the effects of diltiazem, an L-type Ca(2+) channel blocker widely used for the control of the ventricular rate in patients with supraventricular arrhythmias, on hKv1.5 and Kv4.3 channels that generate the cardiac ultrarapid delayed rectifier (I(Kur)) and the 4-aminopyridine sensitive transient outward (I(to)) K(+) currents, respectively. hKv1.5 and Kv4.3 channels were stably and transiently expressed in mouse fibroblast and Chinese hamster ovary cells, respectively. Currents were recorded using the whole-cell patch clamp. Diltiazem (0.01 nM-500 muM) blocked hKv1.5 channels, in a frequency-dependent manner exhibiting a biphasic dose-response curve (IC(50)=4.8+/-1.5 nM and 42.3+/-3.6 muM). Diltiazem delayed the initial phase of the tail current decline and shifted the midpoint of the activation (Vh=-16.5+/-2.1 mV vs -20.4+/-2.6 mV, P<0.001) and inactivation (Vh=-22.4+/-0.7 mV vs. -28.2+/-1.9 mV, P<0.001) curves to more negative potentials. The analysis of the development of the diltiazem-induced block yielded apparent association (k) and dissociation (P) rate constants of (1.6+/-0.2) x 10(6) M(-1)s(-1) and 46.8+/-4.8 s(-1), respectively. Diltiazem (0.1 nM-100 muM) also blocked Kv4.3 channels in a frequency-dependent manner exhibiting a biphasic dose-response curve (IC(50)=62.6+/-11.1 nM and 109.9+/-12.8 muM). Diltiazem decreased the peak current and, at concentrations > or =0.1 microM, accelerated the inactivation time course. The apparent association and dissociation rate constants resulted (1.7+/-0.2) x 10(6) M(-1)s(-1) and 258.6+/-38.1 s(-1), respectively. Diltiazem, 10 nM, shifted to more negative potentials the voltage-dependence of Kv4.3 channel inactivation (Vh=-33.1+/-2.3 mV vs -38.2+/-3.5 mV, n=6, Plt;0.05) the blockade increasing at potentials at which the amount of inactivated channels increased. The results demonstrated for the first time that diltiazem, at therapeutic concentrations, decreased hKv1.5 and Kv4.3 currents by binding to the open and the inactivated state of the channels.

A practical kinetic model for efficient isolation of useful antibodies from phage display libraries

To isolate phages displaying a practical and useful antibody with a high k on value and/or a low k off value from phage display antibody libraries, we developed a rational strategy based on a kinetic model. In the model, the recovery of a phage displaying an antibody after a round of biopanning is expressed as a function of five parameters, the apparent association rate constant of the phage antibody to the immobilized antigen ( k on′), the apparent dissociation rate constant of the phage antibody from the immobilized antigen ( k off′), the effective antigen concentration ( C), the time for the binding process ( t b) and the time for the washing process ( t w). An optimum set of operating parameters ( C, t b and t w) for isolating phages displaying an antibody with a high k on value was designed based on the model. Three rounds of biopanning were carried out under the designed conditions, against a phage library in which the hypervariable regions of an original antibody were randomized. All isolated phages displayed an antibody with a higher k on value and one displayed an antibody with a 30-fold greater k on value than that of the original antibody. Experimental conditions which improve the efficiency of conventional off-rate selections are also described.

Binding interaction studies of the immobilized Salmonella typhimurium with extracellular matrix and muscle proteins, and polysaccharides

Our research attempts to understand the real-time interactions of immobilized Salmonella typhimurium with extracellular membrane proteins (collagen I, fibronectin and laminin) and muscle proteins (actin and myosin). Salmonella cells were immobilized on the sensor chip of a surface plasmon resonance (SPR) biosensor. Typical results showed that collagen I and myosin had higher binding responses to the S. typhimurium surface but laminin, actin and fibronectin had lower binding responses. The binding kinetics of collagen I and Salmonella cell surface showed an apparent dissociation and association rate constants of 3.90E−4 s −1 and 1.07E+4 mol −1 s −1. Using the model system developed in our laboratory, the interactions of carrageenans and other polysaccharides with collagen and the Salmonella sensor surface were evaluated. The κ-carrageenans blocked 92–100% binding of collagen to the Salmonella surface, while sodium alginate and low methoxy pectin blocked 50% and 18% binding, respectively. These biosensor studies allowed the rapid evaluation of compounds that may prevent bacterial attachment to poultry skin and carcasses, thus reducing pathogen contamination of poultry foods.

Fluoxetine blocks cloned neuronal A-type K+ channels Kv1.4.

The effects of fluoxetine were studied on cloned K+ channel Kv1.4 stably expressed in Chinese hamster ovary (CHO) cells using the whole-cell configuration of the patch-clamp technique. Extracellular application of various concentrations of fluoxetine inhibited the amplitude of the peak current of Kv1.4 and accelerated its inactivation time course in a concentration-dependent manner. Thus, fluoxetine decreased Kv1.4 (the integral of the outward current) in a concentration-dependent manner; the IC50 was 33.1 +/- 2.5 microM. The inhibitory effect of fluoxetine was time-dependent. The apparent association (k) and dissociation (l) rate constants measured at +40 mV were 3.5 +/- 0.7 microM-1s-1 and 132.5 +/- 13.3 s-1, respectively. The Kd (= l/k) was 37.9 microM, which was close to the value obtained from the concentration-response curve. The block produced by fluoxetine increased steeply between -30 and 0 mV, which corresponded with the voltage range for channel opening. The fluoxetine block was constant at more depolarized potentials, suggesting that the block by fluoxetine was not voltage dependent. Our data indicate that fluoxetine blocks Kv1.4 channels by preferentially binding to open state.

Fluorescence correlation spectroscopy analysis of the dynamics of tubulin interaction with RB3, a stathmin family protein

We have used fluorescence correlation spectroscopy to analyze the interaction of GTP-tubulin with rhodamine-labeled RB3, a neural protein of the stathmin family, and to determine the kinetic pathway of the association process. RB3 displayed slow association–dissociation kinetics with tubulin depending on the square of the tubulin concentration. The values of the apparent association and dissociation rate constants of the complex of two tubulin dimers and RB3 are determined to be (3.52±0.14)×10 −3 μM −2/s and (1.9±0.6)×10 −3 s −1 respectively. The value of the equilibrium dissociation constant for the first tubulin–RB3 interaction is estimated to be ⩾7 μM at 20°C.

Discontinuous residues of factor IX constitute a surface for binding the anti-factor IX monoclonal antibody A-5

Anti-human factor IX monoclonal antibody, A-5 (Mab A-5), has been widely used in structure-function studies for factor IX. Mab A-5 recognizes the catalytic domain of human factor IX (FIX). Regions important for Mab A-5 binding have previously been localized to the amino terminus of the heavy chain of factor IX, encompassing amino acid residues 181–310 [Blood (74) 971]. We have selected 20 positions in this region for alanine-scanning mutagenesis. We found that Mab A-5 failed to react with the recombinant factor IX mutants with substitutions at positions 228 and 252. Mab A-5 also reacted to a lesser extent to FIXD276A (factor IX with alanine substitution for aspartic acid at residue 276) and FIXK201A/D203A (double alanine substitutions at residues 201 and 203). The apparent dissociation rate constants ( K D) in binding Mab A-5 were 6.0×10 −9, 1.4×10 −8 and 2.0×10 −8 M, for wild-type FIX, FIXK201A/D203A and FIXD276A, respectively. The increased K D values of the two FIX mutants are mainly owing to the increased dissociation rates. These affected residues constitute a surface opposite from the factor VIIIa binding surface. We conclude that the epitope for Mab A-5 is on a surface composed of residues 228, 252, 276, and 201 or 203. This surface, which may not be important for factor VIII binding, contains a strong antigenic region on factor IX.

Kinetic analysis of the interactions between Troponin C (TnC) and Troponin I (TnI) binding peptides: evidence for separate binding sites for the ‘structural’ N‐terminus and the ‘regulatory’ C‐terminus of TnI on TnC

The Ca(2+)/Mg(2+)-dependent interactions between TnC and TnI play a critical role in regulating the 'on' and 'off' states of muscle contraction as well as maintaining the structural integrity of the troponin complex in the off state. In the present study, we have investigated the binding interactions between the N-terminus of TnI (residues 1-40 of skeletal TnI) and skeletal TnC in the presence of Ca(2+) ions, Mg(2+) ions and in the presence of the C-terminal regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). Our results show the N-terminus of TnI can bind to TnC with high affinity in the presence of Ca(2+) or Mg(2+) ions with apparent equilibrium dissociation constants of K(d(Ca(2+) ) ) = 48 nM and K(d(Mg(2+) ) ) = 29 nM. The apparent association and dissociation rate constants for the interactions were, k(on) = 4.8 x 10(5) M (-1) s(-1), 3.4 x 10(5) M (-1) s(-1) and k(off) = 2.3 x 10(-2) s(-1), 1.0 x 10(-2) s(-1) for TnC(Ca(2+)) and TnC(Mg(2+)) states, respectively. Competition studies between each of the TnI regions and TnC showed that both TnI regions can bind simultaneously to TnC while native gel electrophoresis and SEC confirmed the formation of stable ternary complexes between TnI(96-139) (or TnI(96-131)) and TnC-TnI(1-40). Further analysis of the binding interactions in the ternary complex showed the binding of the TnI regulatory region to TnC was critically dependent upon the presence of both TnC binding sites (i.e. TnI(96-115) and TnI(116-131)) and the presence of Ca(2+). Furthermore, the presence of TnI(1-40) slightly weakened the affinity of the regulatory peptides for TnC. Taken together, these results support the model for TnI-TnC interaction where the N-terminus of TnI remains bound to the C-domain of TnC in the presence of high and low Ca(2+) levels while the TnI regulatory region (residues 96-139) switches in its binding interactions between the actin-tropomyosin thin filament and its own sites on the N- and C-domain of TnC at high Ca(2+) levels, thus regulating muscle contraction.

Binding of L-arginine and imidazole suggests heterogeneity of rat brain neuronal nitric oxide synthase.

Nitric oxide synthase (NOS) is inhibited by imidazole, which binds to the heme in a low-spin complex absorbing at 428 nm. Conversion by L-arginine of this complex into a high-spin species absorbing at 395 nm is a common method to determine the binding parameters of Arg. However, both Arg-competitive and noncompetitive inhibition of NOS by imidazole has been reported, and optical studies with neuronal NOS provided no evidence for imidazole affecting Arg binding. We investigated the cause for these paradoxical observations with recombinant rat brain neuronal NOS. Imidazole bound to nNOS with a K(d)(app) of 50 microM; tetrahydrobiopterin (BH4) lowered the affinity of nNOS for imidazole 4-fold. The enzyme behaved heterogeneously with respect to Arg binding. Most of nNOS (65-80%) showed competition between Arg and imidazole. In the presence of BH4, a K(d)(Arg) of 1 microM could be estimated for this fraction, as well as apparent association and dissociation rate constants of 2.5 x 10(6) M(-1) x s(-1) and 2.5 s(-1). A second fraction of nNOS (20-30%) exhibited little or no competition. Consequently, Arg binding did not cause dissociation of the imidazole complex for this fraction, and complete generation of the high-spin state by Arg could not be achieved in the presence of imidazole. A third fraction (< or =10%) bound Arg with low affinity (K(d) 1-2 mM). Because of this heterogeneity, titration curves with Arg became almost uninterpretable. We propose that this heterogeneous response of nNOS toward Arg and imidazole is underlying the apparently conflicting results reported in the literature.

Adsorption kinetics of β-lactoglobulin on a polyclonal immunochromatographic support

β-Lactoglobulin is one of the main components of whey proteins. Among other reasons, its allergenicity makes its determination in hypoallergenic foods and bio-pharmaceutical products necessary. Immunoaffinity chromatography is a widely accepted technique for purification and analysis of proteins. Knowledge of the apparent kinetics of the adsorption of β-lactoglobulin onto the anti-β-lactoglobulin immunochromatographic column is important to optimize the analytical process. High-performance frontal affinity chromatography was used to study the apparent kinetics of the adsorption process. Langmuir and bi-Langmuir kinetic models, assuming one and two kinds of binding sites, respectively, were used to characterize the adsorption kinetics of β-lactoglobulin B on a polyclonal immunoadsorbent. Very good fits were obtained with the bi-Langmuir model for two different concentrations of β-lactoglobulin and this allowed us to calculate the apparent adsorption rate constants and the column capacities for both kinds of sites. Experimental results indicate the possibility that the adsorption process is not irreversible. The values of the apparent dissociation rate constants leading to the best fit were estimated and the affinity constants were calculated.

In vivo kinetic analysis of covalent binding between N-acetyl-L-cysteine and plasma protein through the formation of mixed disulfide in rats.

This investigation was undertaken to study the relationship between plasma drug clearance and covalent protein-binding kinetics of N-acetyl-L-cysteine (NAC). NAC was intravenously administered to rats via a bolus injection or continuous infusion. Plasma concentrations of protein-unbound and total NAC were analyzed using a compartment model, taking into consideration of the protein binding process, and the apparent first-order binding and dissociation rate constants (kon and koff) were obtained. Plasma total NAC after a bolus injection showed biphasic elimination with an inflection point at 1 hr. After 1 hr, NAC was largely present in the covalent protein-bound form. During the steady state of the infusion, approximately 30%-40% of plasma NAC bound with protein covalently. The kon, koff, and the elimination rate constant of protein-unbound drug (ke) were 0.23, 0.57, and 4.3 hr(-1). The dissociation half-life of NAC from protein estimated from koff was in agreement with the elimination half-life of plasma total NAC. This suggests that the dissociation of NAC from protein rate-limited the drug elimination in plasma (koff < ke). We demonstrated that plasma total drug clearance is kinetically limited by covalent protein binding. The compartmental model described here is useful for analyzing its kinetics in vivo.

Real-time Monitoring of the Interactions of Transforming Growth Factor-β (TGF-β) Isoforms with Latency-associated Protein and the Ectodomains of the TGF-β Type II and III Receptors Reveals Different Kinetic Models and Stoichiometries of Binding

Mature transforming growth factor-beta (TGF-beta) is proteolytically derived from the C terminus of a precursor protein. Latency-associated protein (LAP), the N-terminal remnant of the TGF-beta precursor, is able to bind and neutralize TGF-beta. Mature TGF-beta exerts its activity by binding and complexing members of two subfamilies of receptors, the type I and II receptors. In addition to these signaling receptors, TGF-beta can also interact with an accessory receptor termed the type III receptor. Using a surface plasmon resonance-based biosensor (BIAcore), we determined the mechanisms of interaction of four binding proteins (LAP, the type II and III receptor ectodomains (EDs), and a type II receptor ED/Fc chimera) with three TGF-beta isoforms, and we quantified their related kinetic parameters. Using global fitting based on a numerical integration data analysis method, we demonstrated that LAP and the type II receptor/Fc chimera interacted with the TGF-beta isoforms with a 1:1 stoichiometry. In contrast, the type II ED interactions with TGF-beta were best fit by a kinetic model assuming the presence of two independent binding sites on the ligand molecule. We also showed that the type III ED bound two TGF-beta molecules. Further experiments revealed that LAP was able to block the interactions of TGF-beta with the two EDs, but that the two EDs did not compete or cooperate with each other. Together, these results strongly support the existence of a cell-surface complex consisting of one type III receptor, two TGF-beta molecules, and four type II receptors, prior to the recruitment of the type I receptor for signal transduction. Additionally, our results indicate that the apparent dissociation rate constants are more predictive of the neutralizing potency of these TGF-beta-binding proteins (LAP, the type II and III receptor EDs, and the type II receptor/Fc chimera) than the apparent equilibrium constants.

Deconstructing the interaction of glu-plasminogen with its receptor α-enolase

Objective: Plasminogen binds with apparent low affinity to cell-surface receptors via its lysine binding sites. This enhances/stabilizes the activation-susceptible conformation. However, it is not known whether this lysine-mediated conformational change of plasminogen may affect its subsequent dissociation rate and hence its stability at the cell surface. Therefore, we sought to determine the relationship between the lysine-dependent conformation of plasminogen and its dissociation rate from its receptor.Design: BIACORE experiments were used to determine the kinetics of the interaction of glu-plasminogen with its receptor α-enolase. Intrinsic and extrinsic fluorescence spectroscopy were utilized to confirm if α-enolase induced a conformational change to glu-plasminogen as predicted by analyses of the BIACORE data.Results: The dissociation of glu-plasminogen from α-enolase was mediated by at least two components with apparent dissociation rate constants of kd1= 4.7 × 10−2s−1and kd2= 1.6 × 10−3s−1. This second slower dissociation event reflects an increase in the stability of the complex. Global analysis of the interaction suggested a two-state conformational change reaction, mediated by a concentration-dependent increase in the initial association rate constant. The apparent Kdpredicted by this analysis was 1μM. Fluorescence spectroscopy confirmed that α-enolase induced a more open conformation of glu-plasminogen.Conclusions: These results provide direct evidence that the binding of glu-plasminogen to α-enolase is not simply a low-affinity interaction, but involves a multivalent, competition binding reaction that is associated with a glu-plasminogen conformational change. This mechanism is compatible with the structure of glu-plasminogen. This has implications for the stability of binding and activation of glu-plasminogen at the cell surface.

Application of Surface Plasmon Resonance toward Studies of Low-Molecular-Weight Antigen–Antibody Binding Interactions

Methods for studying low-molecular-weight antigen–antibody binding interactions using surface plasmon resonance detection are presented. The experimental parameters most relevant to studies of low-molecular-weight antigen–antibody binding interactions are discussed. Direct kinetic analysis of the binding interactions is most informative, providing both apparent association and dissociation rate constants from which equilibrium constants can be calculated. Equilibrium analysis, including steady-state and solution affinity studies, offers an alternative approach to direct kinetic analysis when knowledge of the individual kinetic rate constants is not required or difficult to determine. The various methods are illustrated by studies of an anti-T4 Fab fragment binding interaction with several thyroxine analogs. The methods utilized were dependent on the affinity of the interaction. The high-affinity anti-T4 Fab fragment/l-T4 binding interaction was evaluated using direct kinetic analysis. An intermediate affinity anti-T4 Fab fragment/l-T3 binding interaction was evaluated using a combination of direct kinetic analysis, steady-state analysis, and solution affinity analysis. The relatively weak anti-T4 Fab fragment/l-T2 binding interaction was evaluated using steady-state and solution affinity analysis protocols. Several thyroxine tracers that could not be immobilized to a biosensor surface were also evaluated via the solution affinity format. In cases where a given binding interaction was examined using multiple methods the results were comparable.

Changes in apparent rates of receptor binding in the intact brain in relation to the heterogeneity of reaction environments.

Neuroreceptor imaging by PET or SPECT has been widely applied in the field of neurobiology, from basic to clinical investigations, and has the potential to reveal the neurochemical basis of various neurological and psychiatric diseases as well as to provide new knowledge in the field of neuropharmacology. In contrast to the static nature of in vitro systems, neurotransmission systems in the intact brain constitute part of a dynamic and communicating environment. Thus, it is important to develop new functional imaging methods that reflect neural communications and the dynamism of signal transmission in the living brain. In vivo receptor binding can be altered not only by competitive inhibition by endogenous neurotransmitters but by trans-synaptic effects, and investigation of neural interactions by detection of changes in receptor binding therefore presents a potential method for studying this phenomenon. Recently, several PET studies on in vivo neural interactions using the D2 receptor ligand [11C]-raclopride concluded that the phenomenon was mediated by changes in synaptic endogenous dopamine concentrations that compete with [11C]-raclopride binding for neuroreceptor occupancy. However, a growing body of evidence indicates that these changes in in vivo receptor binding cannot be fully explained by competitive inhibition by endogenous ligand, and alternative mechanisms for the interneuronal modulation of receptor binding are addressed. This review highlights some of the discrepancies observed between in vitro and in vivo receptor binding studies with respect to a number of phenomena, including the heterogeneity of the reaction field surrounding receptors. Quantitative receptor binding studies are usually analyzed by using 'static' binding parameters, such as the Bmax, and KD, which are normally determined by in vitro assays. In addition to these parameters, the apparent association and dissociation rate constants (kon, koff) play equally significant roles in receptor binding in the intact brain is expected. The concepts of "diffusion boundary" and "reaction volume" are introduced, and discussions on some of the discrepancies between in vivo and in vitro receptor binding phenomena are presented.

Effects of propafenone and 5-hydroxy-propafenone on hKv1.5 channels.

1. The goal of this study was to analyse the effects of propafenone and its major metabolite, 5-hydroxy-propafenone, on a human cardiac K+ channel (hKv1.5) stably expressed in Ltk- cells and using the whole-cell configuration of the patch-clamp technique. 2. Propafenone and 5-hydroxy-propafenone inhibited in a concentration-dependent manner the hKv1.5 current with K(D) values of 4.4+/-0.3 microM and 9.2+/-1.6 microM, respectively. 3. Block induced by both drugs was voltage-dependent consistent with a value of electrical distance (referenced to the cytoplasmic side) of 0.17+/-0.55 (n=10) and 0.16+/-0.81 (n=16). 4. The apparent association (k) and dissociation (l) rate constants for propafenone were (8.9+/-0.9) x 10(6) M(-1) s(-1) and 39.5+/-4.2 s(-1), respectively. For 5-hydroxy-propafenone these values averaged (2.3+/-0.3) x 10(6) M(-1) s(-1) and 21.4+/-3.1 s(-1), respectively. 5. Both drugs reduced the tail current amplitude recorded at -40 mV after 250 ms depolarizing pulses to +60 mV, and slowed the deactivation time course resulting in a 'crossover' phenomenon when the tail currents recorded under control conditions and in the presence of each drug were superimposed. 6. Both compounds induced a small but statistically significant use-dependent block when trains of depolarizations at frequencies between 0.5 and 3 Hz were applied. 7. These results indicate that propafenone and its metabolite block hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner and the concentrations needed to observe these effects are in the therapeutical range.

Dissociation Rate Kinetics in a Solid-Phase Flow Immunoassay

Solid-phase displacement assays allow extremely fast analyses when performed under continuous flow conditions. Continuous dissociation of labeled antigen from the immobilized saturated antibodies occurs even in the absence of competing unlabeled antigen. This spontaneous dissociation creates more unoccupied antibody binding sites which affect the magnitude of the signal generated. In order to evaluate the impact of this phenomenon in more detail, we extended the law of mass action to solid-phase binding assays and analyzed the dissociation kinetics of labeled antigen under continuous flow conditions. The effect of the flow on the dissociation kinetics was determined by calculation of the apparent dissociation rate constants (kd) which increase with an increase in the flow rate. These dissociation rate constants are approximately 20- to 30-fold lower than those obtained from displacement studies (i.e., in the presence of competing unlabeled antigen). The difference in the dissociation rate constants obtained in the two studies is most likely a function of the degree of reassociation. The results of this study provide a basis for better understanding antibody kinetics at solid-liquid interfaces under flow conditions.