Characterization of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)adenosine 5'-triphosphate as a fluorescent probe of the ATP site of sodium and potassium transport adenosine triphosphatase. Determination of nucleotide binding stoichiometry and ion-induced changes in affinity for ATP

The fluorescent ATP derivative 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate (TNP-ATP) binds specifically with enhanced fluorescence to the ATP site of purified eel electroplax sodium-potassium adenosine triphosphatase, (Na,K)-ATPase. A single homogeneous high affinity TNP-ATP binding site with a KD of 0.04 to 0.09 microM at 3 degrees C and 0.2 to 0.7 microM at 21 degrees-25 degrees C was observed in the absence of ligands when binding was measured by fluorescence titration or with [3H]TNP-ATP. ATP and other nucleotides competed with TNP-ATP for binding with KD values similar to those previously determined for binding to the ATP site. Binding stoichiometries determined from Scatchard plot intercepts gave one TNP-ATP site/175,000 g of protein (range: 1.64 X 10(5) to 1.92 X 10(5) when (Na,K)-ATPase protein was determined by quantitative amino acid analysis. The ratio of [3H]ouabain sites to TNP-ATP sites was 0.91. These results are inconsistent with "half-of-sites" binding and suggest that there is one ATP and one ouabain site/alpha beta protomer. (Na,K)-ATPase maintained a high affinity for TNP-ATP regardless of the ligands present. K+ increased the KD for TNP-ATP about 5-fold and Na+ reversed the effect of K+. The effects of Na+, K+, and mg2+ on ATP binding at 3 degrees C were studied fluorimetrically by displacement of TNP-ATP by ATP. The results are consistent with competition between ATP and TNP-ATP for binding at a single site regardless of the metallic ions present. The derived KD values for ATP were : no ligands, 1 microM; 20 mM NaCl, 3-4 microM; 20 mM KCl, 15-19 microM; 20 mM Kcl + 4 mM MgCl2, 70-120 microM. These results suggests that a single ATP site exhibits a high or low affinity for ATP depending on the ligands present, so that high and low affinity ATP sites observed kinetically are interconvertible and do not co-exist independently. We propose that during turnover the affinity for ATP changes more than 100-fold owing to the conformational changes associated with ion binding, translocation, and release.     

Characterization of the binding of the fluorescent ATP analog TNP-ATP to insulysin

It has recently been reported that insulin-degrading enzyme (IDE) contains an allosteric site which binds polyanions such as ATP and PPPi. This site is distinct from the catalytic site where homotrophic allosteric effects are produced. In this study, we have characterized the binding of ATP to this anion binding site using the fluorescent ATP analog 2',3'-O-(2,4,6-trinitrophenyl)-adenosine triphosphate (TNP-ATP), which exhibits a higher affinity to the enzyme than ATP itself. TNP-ATP binding to IDE was accompanied by a more than 4-fold increase in fluorescence. The dissociation constant (K(D)) of TNP-ATP was determined as 1.15 microM, while the activation constant (K(A)) was determined to be 1.6 microM. Competition experiments were used to show that ATP (Ki = 1.3 mM) and PPPi (Ki = 0.9mM) bind with a higher affinity than ADP (2.2 mM) and AMP (4.0 mM). Adenosine did not bind to the anion binding site.     

The dnaB protein of Escherichia coli: mechanism of nucleotide binding, hydrolysis, and modulation by dnaC protein

The mechanism of nucleotide binding and hydrolysis by dnaB protein and dnaB X dnaC protein complex has been studied by using fluorescent nucleotide analogues. Binding of trinitrophenyladenosine triphosphate (TNP-ATP) or the corresponding diphosphate (TNP-ADP) results in a blue shift of the emission maximum and a severalfold amplification of the fluorescence emission of the nucleotide analogues. Scatchard analysis of TNP-ATP binding indicates that TNP-ATP binds with a high affinity (Kd = 0.87 microM) and a 8.5-fold enhancement of fluorescence emission of the nucleotide. Only three molecules of TNP-ATP or TNP-ADP bind per hexamer of dnaB protein in contrast to six molecules of ATP or ADP binding to a dnaB hexamer. TNP-ATP and TNP-ADP are both competitive inhibitors of single-stranded (SS) DNA-dependent ATPase activity of dnaB protein. TNP-AMP neither binds to dnaB protein nor inhibits the ATPase activity. Formation of dnaB X dnaC complex by dnaC protein results in diminution of the TNP-ATP fluorescence enhancement and a concomitant decrease in the SS DNA-dependent ATPase activity. Kinetic analysis of the ATPase activity of dnaB X dnaC complex indicates that the decrease in the ATPase activity on complex formation is due to a reduction of the maximal velocity (Vmax). The dnaB protein hydrolyzes both TNP-ATP and dATP, however, with an extremely slow rate in the presence of single-stranded M13 DNA. The 2'-OH group of the nucleotide most likely plays an important role in the hydrolysis reaction but not in the nucleotide binding.     

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.     

Human aldehyde dehydrogenase: coenzyme binding studies

The binding of NADH and NAD+ to the human liver cytoplasmic, E1, and mitochondrial, E2, isozymes at pH 7.0 and 25 degrees C was studied by the NADH fluorescence enhancement technique, the sedimentation technique, and steady-state kinetics. The binding of radiolabeled [14C]NADH and [14C]NAD+ to the E1 isozyme when measured by the sedimentation technique yielded linear Scatchard plots with a dissociation constant of 17.6 microM for NADH and 21.4 microM for NAD+ and a stoichiometry of ca. two coenzyme molecules bound per enzyme tetramer. The dissociation constant, 19.2 microM, for NADH as competitive inhibitor was found from steady-state kinetics. With the mitochondrial E2 isozyme, the NADH fluorescence enhancement technique showed only one, high-affinity binding site (KD = 0.5 microM). When the sedimentation technique and radiolabeled coenzymes were used, the binding studies showed nonlinear Scatchard plots. A minimum of two binding sites with lower affinity was indicated for NADH (KD = 3-6 microM and KD = 25-30 microM) and also for NAD+ (KD = 5-7 microM and KD = 15-30 microM). A fourth binding site with the lowest affinity (KD = 184 microM for NADH and KD = 102 microM for NAD+) was observed from the steady-state kinetics. The dissociation constant for NAD+, determined by the competition with NADH via fluorescence titration, was found to be 116 microM. The number of binding sites found by the fluorescence titration (n = 1 for NADH) differs from that found by the sedimentation technique (n = 1.8-2.2 for NADH and n = 1.2-1.6 for NAD+).(ABSTRACT TRUNCATED AT 250 WORDS)     

Validation of a new system for androgen binding protein measurement

A new technique that permits measurement of Androgen-Binding Protein (ABP) is validated by reproducibility, linearity and correlation studies. Using this apparatus allowing Scatchard plot analysis, it is also possible to measure association and dissociation rate constants. In addition, it is a very useful tool for a rapid screening of ABP binding capacity during a chromatographic stepwise purification.     

Evidence for muscarinic cholinergic receptors in dog portal vein: binding of [3H]quinuclidinyl benzilate

Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [3H]quinuclidinyl benzilate (QNB) as a ligand. Specific [3H]QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (KD = 15.5 +/- 2.8 pM) with a Bmax of 110 +/- 14.7 fmol/mg protein. Scatchard and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 X 10(9) M-1 min-1 and 0.016 min-1, respectively, were calculated. The dissociation constant calculated from the equation KD = K-1/K+1 was 8.3 pM, such being in good agreement with the Scatchard estimate of KD (15.5 pM). Specific binding of [3H]QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, alpha-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [3H]QNB binding at 10 microM. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.     

A method for determining binding kinetics applied to thiamine-binding protein

A rapid and simple method for assaying the binding activity of thiamine-binding protein is described. By this assay method, the binding characteristics of rice bran thiamine-binding protein have been evaluated with [14C]thiamine as ligand. Analysis of these data by Scatchard plot resulted in linear plots giving a dissociation constant (Kd) for thiamine of 0.55 microM and a maximum binding (Bmax) of 14.5 pmol of ligand bound/microgram of protein. Thiamine binding to the binding protein was time dependent and reached equilibrium at approximately 20 min. The Kob was 0.18 min-1 and the k1 was 1.25 X 10(5) min-1 M-1. Reversibility of thiamine binding at equilibrium was completed at 60 min with a k2 value of 0.052 min-1. The Kd calculated from the reverse rate constant was 0.42 microM. These results indicated that this binding assay method was substantially reliable and accurate.     

Time-resolved fluorescence of bacteriophage Pf1 DNA-binding protein. Determination of oligonucleotide and polynucleotide binding parameters

The binding of oligonucleotides and polynucleotides to the Pf1 DNA-binding protein was followed by fluorescence spectral shift and lifetime measurements, which gave an anomalous value for the stoichiometry of binding. The anomaly was investigated in detail using fluorescence depolarisation to measure the aggregation during the titration and showed that all the fluorescence parameters are related to the specific aggregation of dimers on ligand binding. At saturation, complexes of the protein with the octanucleotide d(GCGTTGCG) and the hexadecanucleotide (dT)16 have rotational correlation times, phi, of 50 ns and 85 ns, corresponding to protein tetramers and octamers, respectively. In the presence of the tetranucleotide d(CGCA) the protein remains as the native dimer (phi = 19 ns). The titration curves could be analysed in terms of two non-equivalent binding sites, with binding constants K1 and K2. Comparison of K1 values for oligonucleotide binding leads to an estimated (single-site) intrinsic binding constant Kint approximately equal to 3 X 10(4) M-1 and a cooperativity parameter omega approximately equal to 100, in agreement with the apparent binding constant Kapp approximately equal to 3 X 10(6) M-1 for polynucleotides. Binding to the second site on the protein dimer is greatly reduced and cannot be determined accurately. The results suggest that the protein dimers bind cooperatively by lateral association along the DNA and that occupation of only one of the two DNA-binding sites of the protein dimers is sufficient to stabilize the nucleoprotein complexes.     

中华蜜蜂化学感受蛋白AcerCSP3的配基结合功能分析

为研究中华蜜蜂Apis cerana cerana化学感受蛋白AcerCSP3在化学感受系统中的生理功能, 本实验通过对AcerCSP3进行原核表达、分离纯化后, 利用荧光法研究了体外重组AcerCSP3与1-NPN以及候选化学配基的结合特征。Scatchard方程显示AcerCSP3与1-NPN的解离常数K_D为8.29 μmol/L, 结合位点数约等于1。在候选配基竞争1-NPN与AcerCSP3结合的实验中, 5种配基均能在200 μmol/L浓度下使1-NPN的相对荧光强度下降至50%以下, 其中β-紫罗兰酮甚至能使1-NPN的相对荧光强度下降至10%左右, 表明候选配基均与AcerCSP3有较强的结合能力, 而3, 4-二甲基苯甲醛与中蜂AcerCSP3的结合能力最强, K_D达到18.77 μmol/L。本研究所用化学配基均为植物花与叶片的挥发性的次生代谢产物, 表明AcerCSP3可能作为中蜂化学感受系统的一部分, 在其搜寻某些植物花粉蜜源时作为气味分子运载体发挥一定的作用。     

An error in interpretation of double-reciprocal plots and Scatchard plots in studies of binding of fluorescent probes to proteins,and alternative proposals for determining binding parameters

One of the objects of experiments in which a fluorochrome is added to suspensions of cell membranes is to determine the parameters n and K _D , the capacity of unit mass of protein to bind fluorochrome and the dissociation constant, respectively. Currently, these are estimated from Scatchard plots, construction of which first requires that observed fluorescence intensity be converted to moles of bound fluorochrome. This in turn is said to be possible by analysis of the intercept of a plot of reciprocal fluorescence intensity against reciprocal protein concentration. However, analysis of the classical mass action equilibrium equation, upon which the foregoing procedures are said to be based, reveals that the intercept of the double-reciprocal plot always underestimates the desired value. The error is formalized and shown to increase without bound with fluorochrome concentration. The error in turn leads to erroneous assessment of n and K _D . Alternative methods for calculating the desired parameters are proposed, based on direct plots of fluorescence intensity.     

Initial binding process of the membrane insertase YidC with its substrate Pf3 coat protein is reversible

The binding of the inner membrane insertase YidC from Escherichia coli to its substrate, the Pf3 coat protein, was examined in vitro by fluorescence spectroscopy. Purified YidC protein was solubilized with the lipid-like detergent n-dodecylphosphocholine and noncovalently labeled with 1-anilino-naphthalene-8-sulfonate (ANS), whereas the Pf3 coat protein was kept in solution by the addition of 10% (v/v) isopropanol to the buffer. The binding of Pf3 coat protein was analyzed by fluorescence quenching of ANS bound to YidC. All binding curves showed a strict hyperbolic form at pH values between 9.0 and 5.0, indicating a reversible and noncooperative binding between YidC and its substrate. Analysis of the data revealed a dissociation constant K D for the binding process in the range of 1 microM. The pH profile of the K D values suggests that the binding of the Pf3 coat protein is dominated by hydrophobic interactions. The titration experiments provide strong evidence for a conformational change of the insertase upon binding a Pf3 coat protein molecule.     

Analysis of binding interaction between the natural apocarotenoid bixin and human serum albumin by circular dichroism and fluorescence spectroscopy

Bixin is an important, pharmacologically active dietary cis-carotenoid, but its interaction with potential macromolecular targets is completely unexplored. This work was aimed to study the binding of bixin to human serum albumin (HSA), the most abundant protein in blood plasma. Circular dichroism (CD) spectroscopy in combination with UV/VIS absorption spectroscopy and fluorescence quenching techniques were applied. Appearance of induced CD bands in the UV- and VIS-absorption spectral regions indicated the formation of non-covalent carotenoid-albumin complexes. Shape and spectral position of the extrinsic Cotton effects suggested the binding of a single bixin molecule to HSA in chiral conformation. Scatchard and non-linear regression analyses of CD titration data resulted in similar values for the association constant (Ka = 6.6 and 4.6x10(5) M(-1), resp.) and for the number of binding sites (n = 1). The binding interaction was independently confirmed by fluorescence-quenching experiment from which the binding parameters were also calculated. CD Displacement measurements performed with marker ligands established that the main drug binding sites of HSA are not involved in binding of bixin. Palmitic acid decreased the amplitude of the induced CD bands suggesting a common albumin binding site for bixin and long-chain fatty acids. The above data indicate that HSA plays a significant role in the plasma transportation of bixin and related dietary carboxylic acid carotenoids.     

Kinetic determination of talin-actin binding

Smooth muscle talin prepared from chicken gizzard binds to skeletal muscle actin in vitro. The stoichiometry of 1:3 for talin:fluorescent labelled G-actin was confirmed by steady state titration and viscosity measurements under non-polymerizing conditions. The binding constant (Kd) of talin and G-actin was determined by continuous fluorescence titration and gave a value of approx 0.3 microM. The association rate constant of talin and fluorescent labelled G-actin of approx 7 x 10(6) M-1 x s-1 was ascertained by the stopped flow method; the dissociation rate constant was calculated at approx 2-3 s-1.     

Ouabain-sensitive 42K binding to Na+, K+-ATPase purified from canine kidney outer medulla.

Specific and ouabain-sensitive potassium binding to Na⁺, K⁺-ATPase was directly observed by centrifugation method with the purified enzyme and ⁴²K. The specific binding reached to saturation level at concentrations more than 0.2 mM KCl and the level was 6.2 nmol per mg ATPase with specific activity of 1470 μmol Pi/h·mg. The binding level, however, was proportional to the enzyme unit used. Simultaneous determination of ⁴²K binding and [³H]ouabain binding showed that two mol of potassium binding were blocked by one mol of ouabain binding per 3.2×10⁵ g enzyme. Although the apparent dissociation constant of the specific potassium binding was estimated at about 50 μM, Scatchard plot of the binding revealed non-linear relationship suggesting that the two potassium sites existed on one catalytic unit of enzyme would be not equivalent but cooperative.     

High affinity binding of paclitaxel to human serum albumin.

Paclitaxel, a very potent antitumor agent is a hydrophobic molecule with low aqueous solubility. Its currently used formula (Taxol) contains the drug in a 1 : 1 (v/v) mixture of ethanol and Cremophor EL. To minimize vehicle-related toxicity, we developed a novel, water-soluble formulation in which paclitaxel is bound noncovalently to human serum albumin. For this purpose, studies of the paclitaxel-albumin binding equilibrium were performed. Paclitaxel dissolved in ethanol was added to the aqueous solution of human serum albumin. Precipitated paclitaxel was removed and unbound drug was separated by ultrafiltration. Paclitaxel concentration was measured by RP-HPLC. Binding data were evaluated based both on the Scatchard plot and the general binding equation describing binding equilibria with the stepwise stoichiometric binding constants. The Scatchard plot was found to be curvilinear with a slight positive slope of the final part. Parameters of high affinity specific binding were determined from the initial part of the curve (nsp = 1.3 and Ksp = 1.7 x 10(6) M(-1)). Stoichiometric binding constants were estimated by fitting the general binding equation to the experimental data (K1 = 2.4 x 10(6) M(-1) and K2 = 1.0 x 10(5) M(-1)). Saturation of the protein with paclitaxel, similarly to other ligands of albumin, could not be reached. The greatest observed value of r (number of paclitaxel molecules bound to one albumin molecule) was 6.6.     

Cyanide binding to canine myeloperoxidase

Equilibria and kinetics of cyanide binding to canine myeloperoxidase were studied. Spectral results support the presence of two heme binding sites; an isosbestic point at 444 nm and a linear Scatchard plot suggest that the binding affinity of cyanide to the two subunits of the enzyme is the same. The dissociation constant is 0.53 microM. The pH dependence of the apparent second order rate constant indicates the presence of an acid-base group on the enzyme with a pKa of 3.8 +/- 0.1. The protonated form of cyanide binds to the basic enzyme with a rate constant of (4.3 +/- 0.3) x 10(6) M-1 s-1.     

Fluorescence competition assay for the assessment of ATP binding to an isolated domain of Na+, K(+)-ATPase

An equation allowing estimation of the dissociation constant for binding of a non-fluorescent ligand to the enzyme is presented that is based on the competitive replacement of the ligand by its fluorescent analog. We derived an explicit formula for the probe fluorescence intensity, which is suitable for nonlinear least-squares analysis. We used this formula to evaluate the binding of ATP to the large cytoplasmic loop of Na+,K(+)-ATPase. The estimated value of KD (6.2+/- 0.7 mM) is comparable with the results from other laboratories for similar constructs obtained by a different method.     

Inhibition of ATP binding to the carboxyl terminus of Kir6.2 by epoxyeicosatrienoic acids

Epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid (AA), are potent and stereospecific activators of cardiac ATP-sensitive K(+)(K(ATP)) channels. EETs activate K(ATP) channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to K(ATP) channel protein. A fluorescent ATP analog, 2,4,6-trinitrophenyl (TNP)-ATP, which increases its fluorescence emission significantly upon binding with proteins, was used for binding studies with glutathione-S-transferase (GST) Kir6.2 fusion proteins. TNP-ATP bound to GST fusion protein containing the C-terminus of Kir6.2 (GST-Kir6.2C), but not to the N-terminus of Kir6.2, or to GST alone. 11,12-EET (5 muM) did not change TNP-ATP binding K(D) to GST-Kir6.2C, but B(max) was reduced by half. The effect of 11,12-EET was dose-dependent, and 8,9- and 14,15-EETs were as effective as 11,12-EET in inhibiting TNP-ATP binding to GST-Kir6.2C. AA and 11,12-dihydroxyeicosatrienoic acid (11,12-DHET), the parent compound and metabolite of 11,12-EET, respectively, were not effective inhibitors of TNP-ATP binding to GST-Kir6.2C, whereas the methyl ester of 11,12-EET was. These findings suggest that the epoxide group in EETs is important for modulation of ATP binding to Kir6.2. We conclude that EETs bind to the C-terminus of K(ATP) channels, inhibiting binding of ATP to the channel.     

Phenylmethylsulfonyl fluoride (PMSF) inhibits nerve growth factor binding to the high affinity (type I) nerve growth factor receptor

Dorsal root ganglia were extirpated from 9-day old embryonic chickens and solubilized in phosphate buffered saline containing 0.5% Noniodet P 40 detergent. When nerve growth factor binding studies are performed on these samples, the expected curvilinear Rosenthal (Scatchard) plot is obtained. However, when the solubilized cell sample is made 1-2 mM in phenylmethylsulfonyl fluoride and nerve growth factor binding is determined, a linear Rosenthal (Scatchard) plot is obtained. The equilibrium dissociation constant obtained from the slope of the line is 1.9 X 10(-9) M, identical to the equilibrium dissociation constant of the low affinity receptor. A similar phenomenon is observed when rat pheochromocytoma cells are solubilized in the non-ionic detergent and nerve growth factor binding is determined. No high affinity binding can be detected for either cell type when detergent solubilized cells are incubated with phenylmethylsulfonyl fluoride.