Binding of acetylcholine and related compounds to purified acetylcholine receptor from Torpedo Californica electroplax

The binding properties of the purified acetylcholine receptor from $\text{Torpedo}\text{californica}$ were investigated. One type of binding was observed for acetylcholine (K_D = 2.3 μM), dimethyl tubocurarine (K_D = 6.2 μM), and decamethonium (K_D = 55 μM). No cooperativity was observed in ligand binding. By virtue of its ligand binding properties, the purified receptor is nicotinic in nature.     

The role of transferrin in the growth of testicular cell lines in serum-free medium

Transferrin (TF) promotes the cell growth of two non-tumorigenic mouse testicular-derived cell lines (TM₁ and TM₃) when cultured in a low-iron serum-free culture medium. No species specificity for growth promotion was observed using mouse, rat, and human TF. Stimulation of growth by apo-TF was biphasic reaching a maximum at 5.0–20.0 μg/ml and declining at higher concentrations. No decrease in TF-stimulatory effect was observed when high doses of diferric TF (Fe-TF) were used, nor when high doses of apo-TF were added in a medium supplemented with additional iron. The decrease in growth-promoting effects of high doses of apo-TF was not restored by the addition of other metals bound by TF indicating that sequestration of these metals was not responsible for the inhibition of growth. The decrease in growth was observed with high levels of apo-TF even in the presence of doses of Fe-TF which elicited a maxima) growth response, supporting the hypothesis that the apo-TF in this system is competing for Fe-TF binding sites and thus diminishing the delivery of Fe to the cells. The growth-promoting effect of apo-TF is blocked when the iron content of the medium is sequestered by Deferoxamine (DFX). However, under these conditions Fe-TF stimulates growth. These observations support the hypothesis that the growth-promoting effect of TF can be accounted for by its role in providing iron to the cells.     

Kinetic regulation of the binding of prothrombin to phospholipid membranes

A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K _D = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K _D values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K _D of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K _D in the 10^?8 M range and (ii) a kinetically rapid, low-affinity interaction with K _D in the 10^?6 M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K _D values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface.     

The role of Mg2+ in the hydrolytic activity of the isolated chloroplast ATPase: Study by high-performance liquid chromatography

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F₁ ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima,K _m values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg²⁺. Although the equations giving the reaction rate are similar in the two cases, the calculated values ofK _m are widely different. The value obtained from the classical mechanism does not agree withK _D , the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na⁺ is more easily explained as a competition between this ion and the activating Mg²⁺, than by the classical mechanism.     

Interaction of three-finger proteins from snake venoms and from mammalian brain with the cys-loop receptors and their models

With the use of surface plasmon resonance (SPR) it was shown that ws-Lynx1, a water-soluble analog of the three-finger membrane-bound protein Lynx1, that modulates the activity of brain nicotinic acetylcholine receptors (nAChRs), interacts with the acetylcholine-binding protein (AChBP) with high affinity, K_D = 62 nM. This result agrees with the earlier demonstrated competition of ws-Lynx1 with radioiodinated α-bungarotoxin for binding to AChBP. For the first time it was shown that ws-Lynx1 binds to GLIC, prokaryotic Cys-loop receptor (K_D = 1.3 μM). On the contrary, SPR revealed that α-cobratoxin, a three-finger protein from cobra venom, does not bind to GLIC. Obtained results indicate that SPR is a promising method for analysis of topography of ws-Lynx1 binding sites using its mutants and those of AChBP and GLIC.     

Specific binding of phorbol ester tumor promoters to mouse skin

The tumor promoter 20-³H-phorbol 12,13-dibutyrate bound in a specific manner to particulate preparations from both whole mouse skin and mouse epidermis. The binding, which was comparable in both whole skin and epidermal preparations, occurred rapidly, was reversible upon addition of non-radioactive ligand and showed high affinity (K_D = 2.4 × 10^?8 M). The potencies of phorbol esters for inhibiting binding of ³H-PDBu corresponded to their biological and tumor-promoting activities: phorbol 12-myristate 13-acetate, K_I = 0.74 nM; phorbol 12,13-didecanoate, K_I = 16 nM; phorbol 12,13-dibenzoate, K_I = 82 nM; mezerein, K_I = 98 nM; phorbol 12,13-diacetate, K_I = 3 μM; phorbol 12,13,20-triacetate, K_I = 5.6 μM; phorbol 13-acetate, K_I = 64 μM. The biologically inactive derivatives phorbol (0.88 mM) and 4α-phorbol 12,13-didecanoate (15 μM) did not inhibit binding. Likewise, ³H-PDBu binding was only weakly inhibited by phorbol-related diterpenes which are highly inflammatory but nonpromoting. These structure-activity relationships suggest that the ³H-PDBu binding activity mediates phorbol ester tumor promotion. ³H-PDBu binding was not inhibited by the nonphorbol promoters examined. Similarly, it was not blocked by compounds which antagonize (dexamethasone acetate, 2 μM; retinoic acid, 10 μM) or mimic (epidermal growth factor, 100 ng/ml; melittin, 25 μg/ml; PGE₂, 1 μM) some of the effects of the phorbol esters in vivo or in vitro.     

Can uranium follow the iron-acquisition pathway? Interaction of uranyl-loaded transferrin with receptor 1

Transferrin receptor 1 (R_D) binds iron-loaded transferrin and allows its internalization in the cytoplasm. Human serum transferrin also forms complexes with metals other than iron, including uranium in the uranyl form (UO₂ ²⁺). Can the uranyl-saturated transferrin (TUr₂) follow the receptor-mediated iron-acquisition pathway? In cell-free assays, TUr₂ interacts with R_D in two different steps. The first is fast, direct rate constant, k ₁ = (5.2 ± 0.8) × 10⁶ M^?1 s^?1; reverse rate constant, k _?1 = 95 ± 5 s^?1; and dissociation constant K ₁ = 18 ± 6 μM. The second occurs in the 100-s range and leads to an increase in the stability of the protein–protein adduct, with an average overall dissociation constant K _d = 6 ± 2 μM. This kinetic analysis implies in the proposed in vitro model possible but weak competition between TUr₂ and the C-lobe of iron-loaded transferrin toward the interaction with R _D.     

Interaction of pyruvate kinase with isatin and deprenyl

The key glycolytic enzyme, pyruvate kinase, exhibits moderate affinity [³H]isatin binding (K_D ～10 μM) which is inhibited by ATP (IC₅₀ 25 μM) and deprenyl (IC₅₀ 5 μM). Interaction of pyruvate kinase with isatin and its inhibition by ATP and deprenyl has also been confirmed using an independent biosensor technique and the immobilized isatin analogue, aminoisatin. This effect has some specificity because the enzyme, creatine phosphokinase, does not exhibit specific isatin-binding. It is suggested that interaction of pyruvate kinase with isatin may reflect some non-glycolytic functions of this enzyme.     

Rational design and molecular engineering of peptide aptamers to target human pancreatic trypsin in acute pancreatitis

Human pancreatic trypsin (hPT) is an established target for acute pancreatitis (AP) therapeutics. Here, a bioinformatics protocol of protein docking, peptide refinement, dynamics simulation and affinity analysis was described to perform rational design and molecular engineering of hPT peptide aptamers. Protein docking was employed to model the intermolecular interactions between hPT and its cognate inhibitory protein, the human pancreatic trypsin inhibitor (hTI). A number of peptide fragments were cut out from the interaction sites of docked hPT–hTI complexes, from which a decapeptide fragment ¹³LNGCTLEYRP²² was found to exhibit potent inhibition against hPT (K _i = 5.3 ± 0.8 μM). We also carried out alanine scanning and virtual mutagenesis to systematically examine the independent contribution of peptide residues to binding affinity, and the harvested knowledge were then used to guide modification and optimization of the decapeptide fragment. Subsequently, inhibition studies of nine promising candidates against recombinant hPT were conducted, from which four samples were successfully identified to have high or moderate potency (K _i < 10 μM). In particular, the peptides LQVCTLEYCN and LQICTLEYCT were found to inhibit hPT activity significantly (K _i = 0.23 ± 0.04 and 0.85 ± 0.18 μM, respectively). Structural analysis of hPT–peptide complex systems unraveled diverse chemical interactions such as hydrogen bonds, salt bridges and hydrophobic forces across the complex interfaces.     

Binding of 1α,25‐dihydroxyvitamin D3 to annexin II: Effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1α,25‐(OH)₂D₃ and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to purified annexin II was inhibited by 1α,25‐(OH)₂D₃ in a concentration‐dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1α,25‐(OH)₂D₃ at 24 μM, 18 μM, and 12 μM and blunted by 6 μM and 3 μM. At a concentration of 12 μM, 1β,25‐(OH)₂D₃ also diminished the binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to annexin II, but cholecalciferol, 25‐(OH)D₃, and 24,25‐(OH)₂D₃ did not. Saturation analyses of the binding of [³H]‐1α,25‐(OH)₂D₃ to purified annexin II showed a K_D of 5.5 × 10⁻⁹ M, whereas [³H]‐1β,25‐(OH)₂D₃ exhibited a K_D of 6.0 × 10⁻⁹ M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration‐dependent effect on [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1α,25‐(OH)₂D₃ binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1β,25‐(OH)₂D₃ to inhibit the binding of [¹⁴C]‐1α,25(OH)₂D₃ bromoacetate to annexin II provides a biochemical explanation for the ability of the 1β‐epimer to inhibit the rapid actions of the hormone in vitro. J. Cell. Biochem. 80:259–265, 2000. © 2000 Wiley‐Liss, Inc.     

Gaba stimulation of 3H-diazepam binding in aged mice

Specific ³H-diazepam binding to washed brain membranes from C57BL/6 mice of different age groups (3, 6, 12, 24 and 36 months) was studied in the absence and presence of 30 μM GABA. GABA treatment was found to be effective in decreasing the K_D of ³H-diazepam binding of approximately 50% in all age groups tested (mean control K_D = 6.5 nM, mean GABA-treated K_D = 3.2 nM). No significant changes with age were observed in benzodiazepine receptor K_D or B_max in the presence or absence of GABA.     

Subunit-subunit interactions in TF1 as revealed by ligand binding to isolated and integrated α and β subunits

The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF₁ and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (K_D(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The K_D(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The K_D(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF₁ binds 2 mol 1-naphthoyl-ATP per mol enzyme with K_D = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 10⁴ M⁻¹s⁻¹. (6) The rate constant for 1-naphthoyl-ATP binding to TF₁ is 6.6 · 10³ M⁻¹ · s⁻¹ (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (k^A₋₁ = 3 · 10⁻³s⁻¹) and a slower second phase (k^A₋₂ < 0.2 · 10⁻³s⁻¹). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF₁ are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.     

Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro

Elevated glutathione transferase (GST) E2 activity is associated with DDT resistance in the mosquito Anopheles gambiae. The search for chemomodulators that inhibit the function of AgGSTE2 would enhance the insecticidal activity of DDT. Therefore, we examined the interaction of novel natural plant products with heterologously expressed An. gambiae GSTE 2 in vitro. Five of the ten compounds, epiphyllocoumarin (Tral-1), knipholone anthrone, isofuranonaphthoquinones (Mr 13/2, Mr13/4) and the polyprenylated benzophenone (GG1) were shown to be potent inhibitors of AgGSTE2 with IC₅₀ values of 1.5 μM, 3.5 μM, 4 μM, 4.3 μM and 4.8 μM respectively. Non-competitive inhibition was obtained for Tral 1 and GG1 with regards to GSH (K_i of 0.24 μM and 0.14 μM respectively). Competitive inhibition for Tral1 was obtained with CDNB (K_i = 0.4 μM) whilst GG1 produced mixed type of inhibition. The K_i and K_i' for GSH for Tral-1 and GG1 were 0.2 μM and 0.1 μM respectively. These results suggest that the novel natural plant products, particularly Tral-1, represent potent AgGSTE2 in vitro inhibitors.     

High affinity ibogaine binding to a mu opioid agonist site

The naturally occurring indole alkaloid ibogaine is of interest because of its reported ability to block drug seeking behavior for extended periods. The compound also potentiates morphine-induced analgesia in mice and reduces certain naltrexone-precipitated withdrawal signs in morphine-dependent rats. Although these results might suggest ibogaine interaction with opioid receptors, previous receptor binding studies (Brain Res. 571:242–247, 1980) found that ibogaine had a K_i value of only 2 μM for the kappa opioid receptor and was virtually inactive in blocking mu and delta receptor binding (K_i >100 μM). The present investigation of ibogaine interaction with the mu opioid receptor from mouse forebrain labeled with [3H]-naloxone, however, yielded significantly more potent mu opioid K_i values. LIGAND analysis indicated that the data were best fit by a two site binding model, with K_i values of about 130 nM and 4 μM, reflecting ibogaine recognition of different agonist affinity states of the receptor. Inclusion of 100 mM NaCl in the assay to induce the agonist low affinity state of the receptor, reduced ibogaine's inhibition of [³H]-naloxone binding. These results suggest that ibogaine is an agonist at the mu opioid receptor with a K_i value of about 130 nM, potentially explaining ibogaine's antinociceptive effects as well as its reported reduction of opioid withdrawal symptoms and attenuation of drug seeking behavior.     

Binding of pyridine coenzymes to the β-subunit of the voltage sensitive potassium channels

The β-subunit of the voltage-sensitive K⁺ channels shares 15–30% amino acid identity with the sequences of aldo–keto reductases (AKR) genes. However, the AKR properties of the protein remain unknown. To begin to understand its oxidoreductase properties, we examine the pyridine coenzyme binding activity of the protein in vitro. The cDNA of K_vβ2.1 from rat brain was subcloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The purified protein was tetrameric in solution as determined by size exclusion chromatography. The protein displayed high affinity binding to NADPH as determined by fluorometric titration. The K_D values for NADPH of the full-length wild-type protein and the N-terminus deleted protein were 0.1±0.007 and 0.05±0.006 M, respectively — indicating that the cofactor binding domain is restricted to the C-terminus, and is not drastically affected by the absence of the N-terminus amino acids, which form the ball and chain regulating voltage-dependent inactivation of the α-subunit. The protein displayed poor affinity for other coenzymes and the corresponding values of the K_D for NADH and NAD were between 1–3 μM whereas the K_D for FAD was >10 μM. However, relatively high affinity binding was observed with 3-acetyl pyridine NADP, indicating selective recognition of the 2′ phosphate at the binding site. The selectivity of K_vβ2.1 for NADPH over NADP may be significant in regulating the K⁺ channels as a function of the cellular redox state.     

Vasoactive intestinal peptide (VIP) binding to solubilized material from rat liver plasma membranes

A non-ionic detergent such as Lubrol-PX extracts in soluble form the VIP-binding structures of rat liver plasma membranes. Detergent-solubitized proteins bind specifically [¹²⁵I]VIP and the complex tracer-protein is identified by the use of Sepharose 6B columns. The interaction is only possible in the absence of detergent (below 0.001%) and is inhibited by native peptide. A molecular weight of about 80,000 was estimated for VIP-binding proteins by reference to a series of globular markers of proteins. Binding to VIP soluble proteins is specific and dependent on time as studied by the Hummel and Dreyer (Biochim. Biophys. Acta 63:530–532, 1962) assay.     

Generation and characterization of biotinylated recombinant Fab antibody fragment against cortisol

This paper describes the in vivo generation method of biotinylated recombinant Fab antibody fragments. The original molecular vectors for Escherichia coli Fab fragments expression were designed. In vivo biotinylated recombinant antibody Fab fragment against cortisol was generated. The kinetic parameters of interaction of these antibody fragments with cortisol-BSA complex were measured via the biolayer interferometry method. An equilibrium dissociation constant of this interaction K_D is 5.48 × 10^–10 M. The interaction is reversible and it could be competitively inhibited by free cortisol addition. These results could be used in generating of immunoassays for quantitative cortisol determinations. The in vivo biotinylation system under review is universal and suitable for expression of any biotinylated Fab fragments in the E. coli system.     

Chronic Antagonist Treatment Does not Alter the Mode of Interaction of Dopamine with Rat Striatal Dopamine Receptors

Abstract

Chronic treatment with the D₁ and D₂ dopamine receptor antagonists SCH 23390 (0.5 mg/kg) and haloperidol decanoate (25 mg/kg) caused an up-regulation in D₁ and D₂ receptor densities, respectively, with no change in K_D. Dopamine (20 μM) interacted with both receptor subtypes in a mixed competitive/non-competitive manner, causing a reduction in ligand binding affinity and an apparent decrease in receptor density. In the presence of dopamine, both vehicle-treated and SCH 23390-treated striatal preparations showed a significant loss in affinity for ³H-SCH 23390 binding to D₁ receptors and a decrease in D₁ receptor density of approximately 26%. Similarly, dopamine caused a substantial loss in ³H-spiperone binding affinity to D₂ receptors and a 46% decrease in B_max in both vehicle-treated and haloperidol-treated membranes. Thus, receptor up-regulation does not appear to alter the mode of interaction of dopamine with rat striatal dopamine receptors.     

Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K_Mox values were around 4-5 μM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK_Mox values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K_Mox was around 1.2 μM for succinate and around 11 μM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K_Mox. However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K_Mox increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.