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PubMedDate not supplied

A new method for monitoring the kinetics of calcium binding to the sarcoplasmic reticulum Ca(2+)-ATPase employing the flash-photolysis of caged-calcium.

The kinetics of Ca2+ binding to the high-affinity sites of the sarcoplasmic reticulum (SR) Ca2(+)-ATPase were directly investigated by continuously monitoring the extravesicular calcium concentration via the metallochromic indicator Arsenazo III following the release of Ca2+ from a photolabile caged-calcium molecule, 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N'-tetrakis [(oxycarbony)methyl]-1,2-ethanediamine (DM-nitrophen), utilizing a pulsed Nd:YAG laser for photolysis. The nature of the binding kinetics is at least biphasic over the first 400 ms for vesicular dispersions of SR. The stoichiometry for calcium binding expressed as Ca:E1 approximately P has been calculated to be approximately 1.4:1 for the pure SR preparation under the reaction conditions employed.

Acetates↗
PubMed1990

Differential analysis of the frequency-dependent effects of class 1 antiarrhythmic drugs according to periodical ligand binding: implications for antiarrhythmic and proarrhythmic efficacy.

The frequency-dependent block of cardiac sodium channels by class 1 antiarrhythmic drugs can be described by a periodical ligand binding process between drug molecules and channel binding sites. This predicts a linear relation between onset-rate constant of frequency-dependent block and diastolic interval as well as saturation of block with high stimulation rates. From both relationships, the binding kinetics (time constant, tau on) and saturation level of block (bdinf) can be estimated. This is exemplified for the frequency-dependent block (reduction of the maximal upstroke velocity of action potentials) induced by prajmaline (10(-6) M). In the same way, the frequency-dependent effects of 11 other class 1 drugs reported in the literature were analyzed and compared with each other. When the drugs are ranked with respect to their binding kinetics (tau on), there is a close relationship to the subclasses (1a, 1b, 1c), with 1b drugs exhibiting the fastest and 1c drugs the slowest kinetics. However, differences also exist in the saturation behavior of frequency-dependent block even within the same subclasses (1a and 1c). Thus, the class 1 drugs can also be subdivided in three other groups exhibiting clearly separated bands of block-frequency relations, with half-maximal saturation occurring at different stimulation rates. Our findings may have differential implications for the antiarrhythmic and proarrhythmic efficacy of class 1 drugs.

Animals↗
PubMed1992

Protein dynamics. An overview on flash-photolysis over broad temperature ranges.

Ligand binding kinetics to heme-proteins between 40 and 300 K point to a regulatory role of protein dynamics. A protein-specific susceptibility of the heme-iron reactivity to dynamic fluctuations emerges from the distribution of reaction enthalpies derived from flash-photolysis measurements below ca. 180 K; we quantify it in terms of 'intramolecular viscosity', postulating that narrow low-temperature enthalpy distributions correspond to low internal viscosity and vice versa. The thermal evolution of ligand binding kinetics suggests, with other results, an interplay between high-frequency transitions of the amino acid side chains and low-frequency collective motions as a possible regulatory mechanism of protein dynamics.

Carbon Monoxide↗
PubMed1989

Effect of plasma protein binding on kinetics of PN 200-110 in the isolated perfused rat heart.

The myocardial accumulation and elimination pharmacokinetics of PN 200-110 (PN) were investigated in the single pass isolated perfused rat heart by two methods. A direct method, radioactivity measurement in myocardial tissue after various perfusion times, and an indirect method, concentration determination in coronary effluent, by fractionary collection of samples, during infusion and elimination periods. Both methods showed that the myocardium could be considered as a one-compartment model with regard to PN pharmacokinetics. The perfusion with a modified Krebs-Ringer (MKR) solution containing 1 nM of (+/-)PN 200-110 and [3H]-(+)PN 200-110 as radioactive tracer, led to an accumulation of about 61.4 fmol.mg-1 myocardial tissue at steady-state. The effect of protein binding on the uptake and pharmacokinetic parameters of PN has been investigated in this isolated perfused heart (IPH) model. binding of PN decreased as a function of increasing bovine serum albumin (BSA) levels in the perfusion solution. As a matter of fact, the mean steady state myocardial concentration of PN was decreased by 42.9, 56.2, 76.5, 83.9 and 95.5% for respectively, 1, 2.5, 6, 10 and 40 g.l-1 of BSA. In the same way, the free fraction, the apparent volume of distribution (Vd) and the distribution and elimination half-lives were decreased. On the contrary, the elimination rate constant was increased.

Animals↗
PubMed1987

Basic kinetics of binding and incorporation with supramolecular aggregates.

Supramolecular particles of spherical shape (such as micelles, vesicles, etc.) are imagined to interact with small (bio)molecules so that either binding or incorporation occurs. This is generally described as an association process whose rate is determined by appropriate diffusion and actual transformation steps. Along these lines pertinent expressions for the phenomenological rate constants are given. In addition, an upper bound for the rate of incorporation has been derived. The results are discussed with respect to their usefulness in practice.

Kinetics↗
PubMed1986

Pituitary binding and internalization of radioiodinated gonadotropin-releasing hormone agonist and antagonist ligands in vitro and in vivo.

In rat pituitary gonadotrophs, the rates of binding and endocytosis of two GnRH superagonist analogs, [D-Ala6,Pro9-NEt]GnRH and [D-Lys6,Pro9-NEt]GnRH, were compared with those of the potent antagonist analog [N-acetyl-D-pCl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]GnRH by quantitative electron microscopic autoradiography. In dispersed pituitary cells, the two agonist analogs showed similar binding kinetics and comparable degrees of sequestration, as measured by their resistance to dissociation by low pH buffer. However, quantification of silver grain localization suggested that cellular internalization of the [D-Ala6]GnRH agonist increased more rapidly than that of the [D-Lys6]GnRH analog. These discrepancies, and the finding that a larger amount of the specifically bound 125I-[D-Ala6]GnRH agonist was removed during glutaraldehyde fixation, indicated that the proportional internalization of this analog was over estimated by quantitative autoradiography owing to loss of cell surface-bound radioligand. We, therefore, employed radioiodinated D-Lys6-substituted analogs to analyze the receptor binding and cellular uptake of GnRH agonist and antagonist derivatives in vivo. After iv injection, a high proportion of the 125I-[D-Lys6]GnRH agonist was translocated into pituitary gonadotrophs within 60 min, whereas the D-Lys6 antagonist was predominantly associated with the plasma membrane during that time. Four hours after injection of the antagonist, an appreciable proportion of silver grains was associated with intracellular organelles, and this trend increased progressively at later time points. The relatively prolonged cellular processing of the GnRH antagonist is consistent with in vivo binding kinetics, and its slower internalization may reflect the basal rate of GnRH receptor turnover in the cell membrane. In addition, the marked difference between the rates of internalization of the bound [D-Lys6]GnRH agonist and antagonist ligands supports the proposal that receptor activation is responsible for the rapid endocytosis of agonist ligands by the GnRH-stimulated pituitary gonadotroph.

Animals↗
PubMedDate not supplied

The influence of transport on the kinetics of binding to surface receptors: application to cells and BIAcore.

Accurate estimation of biomolecular reaction rates from binding data, when ligands in solution bind to receptors on the surfaces of cells or biosensors, requires an understanding of the contributions of both molecular transport and reaction. Efficient estimation of parameters requires relatively simple models. In this review, we give conditions under which various transport effects are negligible and identify simple binding models that incorporate the effects of transport, when transport cannot be neglected. We consider effects of diffusion of ligands to cell or biosensor surfaces, flow in a BIAcore biosensor, and distribution of receptors in a dextran layer above the sensor surface. We also give conditions under which soluble receptors can be expected to compete effectively with surface-bound receptors.

Biological Transport↗
PubMed1985

Kinetics of binding of membrane-active drugs to receptor sites. Diffusion-limited rates for a membrane bilayer approach of 1,4-dihydropyridine calcium channel antagonists to their active site.

Using the model of 1,4-dihydropyridine calcium channel-blocking drug binding to receptors in the cardiac sarcolemmal membrane, diffusion-limited rates of association were calculated for two distinct approaches. In the "aqueous approach," the drug reaches the receptor by diffusion through the bulk solvent, whereas in the "membrane approach," the drug partitions into the membrane bilayer and then diffuses laterally to a specific receptor site. Calculated rates for the membrane approach were approximately 3 orders of magnitude greater than those for the aqueous approach. The membrane approach diffusion-limited rate depends weakly on the sizes of the binding site, the drug molecule, and the vesicle, but depends strongly on ligand asymmetry. Although the measured binding rates for several 1,4-dihydropyridines were all slower than the calculated diffusion-limited rates for either model, other experimental data (such as very high partition coefficients and specific positions of these drugs in the membrane bilayer) suggest that the membrane approach is the most likely. These results have important implications for specifying critical characteristics of active 1,4-dihydropyridines.

Animals↗
PubMed1983

A kinetic and binding study of the reactivity of Escherichia coli ATPase to N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.

Escherichia coli H+-ATPase (ECF1) was inactivated in a time- and concentration-dependent manner by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a selective carboxyl group reagent. Among the subunits of ECF1, only the beta subunit was modified by EEDQ. The reaction of 1 mol of EEDQ per mol of ECF1 resulted in total inactivation, in spite of the fact that the enzyme possesses three beta subunits.

Adenosine Diphosphate↗
PubMed1997

Effect of L-glutamate on cholinergic neurotransmission in various brain regions and during the development of rats, when administered perinatally.

Glutamate, as a monosodium salt (MSG) has neurotoxic effects on some brain regions when systemically given to young rats. Few studies have been conducted to establish the mechanisms involved in studying neurotoxicity resulting in neuronal death by glutamate (Glu) and its effects as related to different brain neuropathologies under in-vivo conditions and where the cholinergic system shows vulnerability. Thus, this paper aims to evaluate the binding kinetics of quinuclynidyl benzylate (QNB) to muscarinic receptors for acetylcholine and the activity of choline acetyltransferase (CAT) in rats treated with MSG (4 mg/g on days 1, 3, 5, and 7 after birth) during the rat development stages (days 14, 21, 30, and 60) in different brain regions. The results show that perinatal treatment with MSG significantly decreases the CAT activity and increases the affinity of [3H]-QNB and the number of receptors of the brain cortex during the ages studied. The striatum showed increased CAT activity and BMAX on days 30 and 60 after birth. Affinity and the number of receptors increased in the hippocampus only between days 21 through 60 after birth. NaCl given at MSG equimolar doses only modified the CAT activity but had no effect on the [3H]-QNB binding kinetics in any of the regions studied. The results show that MSG alters cholinergic neurotransmission in the central nervous system (CNS) and induces the development of compensating events suggesting an involvement in neuronal plasticity during the development of rat CNS.

Animals↗
PubMed1984

Transient kinetics of the binding of ATP to actomyosin subfragment 1: evidence that the dissociation of actomyosin subfragment 1 by ATP leads to a new conformation of subfragment 1.

The initial steps by which ATP dissociates and binds to actomyosin subfragment 1 (acto-SF-1) were studied. Two techniques were used: stopped-flow (for acto-SF-1 dissociation kinetics) and rapid-flow quench with ATP chase quenching (for ATP binding kinetics). The experiments were carried out in 40% ethylene glycol-5 mM KCI, pH 8, at 15 degrees C. Under these conditions, the binding of SF-1 to actin remains very tight. As with SF-1, the ATP chase technique could be used, first, to titrate active sites and, second, to study the kinetics of ATP binding to acto-SF-1. The kinetic constants obtained were compared with those of SF-1 alone and with the acto-SF-1 dissociation kinetics under identical conditions. The kinetics of the acto-SF-1 dissociation did not vary with the actin to SF-1 ratio, but the ATP binding kinetics did, and a maximum value was reached at a mole ratio of 2.5. At high ATP (100 microM), kdiss = 300 s-1, which compares with 49 s-1 and 13 s-1 for the ATP binding kinetics for acto-SF-1 (actin to SF-1 = 1:1) and SF-1, respectively. As with SF-1, the ATP binding to acto-SF-1 follows a hyperbolic law with the ATP concentration. This suggests a rapid equilibrium (K) followed by an essentially irreversible step (k).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1996

Calmidazolium alters Ca2+ regulation of tension redevelopment rate in skinned skeletal muscle.

To examine if the Ca2(+)-binding kinetics of troponin C (TnC) can influence the rate of cross-bridge force production, we studied the effects of calmidazolium (CDZ) on steady-state force and the rate of force redevelopment (ktr) in skinned rabbit psoas muscle fibers. CDZ increased the Ca2(+)-sensitivity of steady-state force and ktr at submaximal levels of activation, but increased ktr to a greater extent than can be explained by increased force alone. This occurred in the absence of any significant effects of CDZ on solution ATPase or in vitro motility of fluorescently labeled F-actin, suggesting that CDZ did not directly influence cross-bridge cycling. CDZ was strongly bound to TnC in aqueous solutions, and its effects on force production could be reversed by extraction of CDZ-exposed native TnC and replacement with purified (unexposed) rabbit skeletal TnC. These experiments suggest that the method of CDZ action in fibers is to bind to TnC and increase its Ca2(+)-binding affinity, which results in an increased rate of force production at submaximal [Ca2+]. The results also demonstrate that the Ca2(+)-binding kinetics of TnC influence the kinetics of ktr.

Actomyosin↗
PubMed1988

Estimation of binding parameters by kinetic data analysis: differentiation between one and two binding sites.

A method that enables the discrimination between binding models and the estimation of binding parameters, based solely on kinetic data, is described. Experimental data from association and dissociation experiments were fitted simultaneously to models with mono- or biphasic kinetics with the aid of a non-linear maximum likelihood computer program. Discrimination between two models can be performed statistically. The protocol was used to study the binding of the antitussive [3H]noscapine to guinea pig brain homogenate. Two binding processes could be discriminated by their kinetics, despite the fact that [3H]noscapine apparently binds to one homogeneous population of binding sites in equilibrium binding experiments. This method might find general application when two populations of binding sites are suspected from kinetic data, but when selective ligands are lacking. Since parameter estimates are obtained independent of equilibrium binding data, our approach could also serve as an independent control of such experiments, with respect to both Kd and Bmax.

Animals↗
PubMed2002

Differences between neonates and adults in carbohydrate sequences and reaction kinetics of plasmin and alpha(2)-antiplasmin.

This study investigates reaction kinetics by slow-binding kinetics methods of both adult and fetal plasmin (Types 1 and 2) with adult and fetal alpha(2)-antiplasmin. In addition, carbohydrate sequences of Fetal and Adult Plasminogen Types 1 and 2, as well as fetal and adult alpha(2)-antiplasmin, were determined by mass spectrometric analysis. All curves of plasmin-alpha(2)-antiplasmin interaction followed the same pattern, indicating reversible slow-binding inhibition with an initial loose complex and a following tight complex. Differences between fetal and adult plasmin reactions with alpha(2)-antiplasmin were predominantly due to the initial loose complex. Values for K(i initial) in the reaction with adult alpha(2)-antiplasmin were 1.5 and 1.6 nM for Fetal Plasmin Types 1 and 2, respectively; compared to 0.3 and 0.7 nM for the corresponding adult types. Increasing concentrations of tranexamic acid resulted in a continuous increase of K(i initial) until a plateau was reached which was similar for all plasmin types. Almost identical values could be obtained when fetal alpha(2)-antiplasmin was used instead of adult alpha(2)-antiplasmin. Mass spectrometric analyses of the glycans present on plasminogen revealed a higher level of truncated N-glycans on the fetal material compared to the adult. The O-glycans of fetal and adult plasminogen were closely similar and only minor differences were observed between N-glycans of fetal and adult alpha(2)-antiplasmin. In conclusion, both fetal plasmin isoforms are less inhibited by alpha(2)-antiplasmin compared to the adult plasmin variants. These findings are important for the understanding of the physiology of the fibrinolytic system in neonates and provide further evidence that differences in glycosylation could be associated with marked effects on protein function.

Adult↗
PubMed2006

Mechanism of intracellular block of the KcsA K+ channel by tetrabutylammonium: insights from X-ray crystallography, electrophysiology and replica-exchange molecular dynamics simulations.

The mechanism of intracellular blockade of the KcsA potassium channel by tetrabutylammonium (TBA) is investigated through functional, structural and computational studies. Using planar-membrane electrophysiological recordings, we characterize the binding kinetics as well as the dependence on the transmembrane voltage and the concentration of the blocker. It is found that the apparent affinity of the complex is significantly greater than that of any of the eukaryotic K(+) channels studied previously, and that the off-rate increases with the applied transmembrane voltage. In addition, we report a crystal structure of the KcsA-TBA complex at 2.9 A resolution, with TBA bound inside the large hydrophobic cavity located at the center of the channel, consistent with the results of previous functional and structural studies. Of particular interest is the observation that the presence of TBA has a negligible effect on the channel structure and on the position of the potassium ions occupying the selectivity filter. Inspection of the electron density corresponding to TBA suggests that the ligand may adopt more than one conformation in the complex, though the moderate resolution of the data precludes a definitive interpretation on the basis of the crystallographic refinement methods alone. To provide a rationale for these observations, we carry out an extensive conformational sampling of an atomic model of TBA bound in the central cavity of KcsA, using the Hamiltonian replica-exchange molecular dynamics simulation method. Comparison of the simulated and experimental density maps indicates that the latter does reflect at least two distinct binding orientations of TBA. The simulations show also that the relative population of these binding modes is dependent on the ion configuration occupying the selectivity filter, thus providing a clue to the nature of the voltage-dependence of the binding kinetics.

Alanine↗
PubMed2004

Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras.

Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical applications, and possibly also treatment applications. Its suggested uses include that of a specific anchor sequence for biologically active peptides to plasmids in a sequence-specific manner. Such complexes, referred to as Bioplex, have already been used to enhance non-viral gene transfer in vitro. To investigate how hybridization of PNAs to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to the same strand of DNA, we have developed a method of quantifying the specific binding of PNA using a PNA labeled with a derivative of the fluorophore thiazole orange (TO). Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNAs, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found increased binding kinetics when two PNAs binding to overlapping sites on opposite DNA strands were used, without the use of chemically modified bases in the PNAs.

Benzothiazoles↗
PubMed1997

Expression, Purification, and Inhibitory Properties of Human Proteinase Inhibitor 8

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.

Journal Article↗
PubMed1982

Interaction of 125I-labeled colicin E1 with Escherichia coli.

Colicin E1 protein was labeled with (125)I to specific activities of up to 2 x 10(8) cpm/mg of protein and with no loss of the colicin biological activity. The labeled colicin bound to colicin E1-sensitive, tolerant, and immune E1-colicinogenic Escherichia coli. An E. coli mutant resistant to colicin E1 exhibited a much lower colicin-binding capacity. The average number of bound colicin molecules per sensitive cell increased as a function of the colicin concentration in the colicin cell interaction mixture and continued to increase even after loss of viability of the entire culture. Up to 2,400 colicin E1 molecules bound per cell, but saturation was not reached. Binding kinetics showed that maximum binding occurred within 2 to 5 min of colicin addition. Survival and binding assays indicated that one colicin killing unit corresponded to an average of about 100 colicin molecules bound per bacterial cell. This number, however, decreased to about 8 in more extensively washed cells. Trypsin digestion of the colicin-treated cells removed the majority of the cell-bound colicin, but in general provided little rescue from colicin killing. At low colicin concentrations, a linear relationship existed between survival and the number of trypsin-inaccessible colicin molecules. Under these circumstances and in agreement with single-hit kinetics, the relationship between the number of colicin killing units and the number of trypsin-inaccessible colicin molecules was close to 1. After trypsin digestion, cells that were nearly saturated with colicin retained about 200 trypsin-inaccessible colicin molecules per cell. The trypsin-inaccessible colicin might represent those colicin molecules that bound to the specific E colicin receptors of E. coli cells.

Adsorption↗