Cooperative monomer binding by polynucleotides. Effect of multiple-loop configurations on formation of triple-stranded complexes

Simulated binding curves for the reaction 2 polymer + monomer = triple-stranded complex are presented, in which loop formation and sliding degeneracy of the polymer adsorption surface are considered. Exact calculations for a polymer chain length N of 11 units suggest that configurations of two or more loops have negligible effect on the isotherm when SW > 1, where S and W are exponential weighting factors for monomer–monomer S and polymer–polymer W nearest neighbor interactions. There is a pronounced effect, however, when SW ? 1. Limiting expressions (N ? 1, but finite) for the single-loop configurations suggest these configurations are negligible at any degree of saturation θ if θ (1 ? θ)^2–k N^3–k ? SW, where k is defined by the weighting factor (j + 1)^?k for a ring of j units. This expression suggests that single monomer-stack configurations are the only significant contributors to the grand partition function at the midpoint of the isotherm if N^3–k ? SW. Furthermore, single-loop configurations are negligible below θ = 0.5 but become dominant above the isotherm midpoint when SW ～ 1 (random binding) if 2 < k < 3. For k > 3 and N → ∞, loop configurations have no effect in any region of the random binding isotherm usually analyzed experimentally (θ < 0.95). Equivalence of matrix and sequence generating methods is also demonstrated.     

Leaf area prediction model for sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) cultivars

In two successive years (2003 and 2004), a set of 16 commercial sugar beet cultivars was established in Randomized Complete Block experiments at two sites in central Greece. Cultivar combination was different between years, but not between sites. Leaf sampling took place once during the growing season and leaf area, LA [cm²], leaf midvein length, L [cm] and maximum leaf width, W [cm] were determined using an image analysis system. Leaf parameters were mainly affected by cultivars. Leaf dimensions and their squares (L², W²) did not provide an accurate model for LA predictions. Using L×W as an independent variable, a quadratic model (y = 0.003 x² − 1.3027 x + 296.84, r ² = 0.970, p<0.001, n = 32) provided the most accurate estimation of LA. With compromises in accuracy, the linear relationship between L×W and LA (y = 0.5083 x + 31.928, r ² = 0.948, p<0.001, n = 32) could be used as a prediction model thanks to its simplicity.     

Spectroscopic investigations on the interactions of potent platinum(II) anticancer agents with bovine serum albumin

The interactions of three platinum(II)-based anticancer complexes [(5,6-dimethyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)]²⁺, [(5,6-dimethyl-1,10-phenanthroline)(1R,2R-diaminocyclohexane)platinum(II)]²⁺, and [(5,6-dimethyl-1,10-phenanthroline)(1,2-diaminoethane)platinum(II)]²⁺ (56MEEN) with BSA have been examined by circular dichroism (CD), fluorescence and ¹H pulsed gradient spin–echo (PGSE) diffusion NMR spectroscopy. The number of association constants and sites differed depending upon the spectroscopic method. This may be because each technique monitors different types of interaction/s and/or as a consequence of the different concentration ranges required for each technique. The titration of BSA with the achiral 56MEEN as monitored by CD indicates a reduction in the α-helical nature of the albumin, with the association constant calculated to be ~5 × 10⁶ M⁻¹ for one site. Due to the chiral nature of the other two complexes, their association with albumin was not monitored using CD but was examined using fluorescence and PGSE diffusion NMR. Titration of BSA with any of the three metal complexes resulted in quenching of fluorescence, with the number of association sites calculated to be ~1.1, with an association constant of ~2 × 10⁵ M⁻¹. PGSE diffusion NMR provided insights into interactions occurring with the BSA in its entirety, rather than with individual regions. Metal complex binding sites were estimated (~10 equivalent) from the diffusion data, with the average association constant for all sites ~10²–10³M⁻¹. These experiments highlight the information that can be elucidated from complementary spectroscopic techniques and demonstrate the usefulness of PGSE diffusion NMR in monitoring multiple weak binding sites, which is of great importance in studying drug-biomolecule interactions.     

Target‐directed screening of the bioactive compounds specifically binding to β2‐adrenoceptor in Semen brassicae by high‐performance affinity chromatography

The bioactive ingredients in Semen sinapis were rapidly screened by immobilized β₂‐adrenoceptor (β₂‐AR) and target‐directed molecular docking. The methods involved the attachment of β₂‐AR using any amino group in the receptor, the simultaneous separation and identification of the retention compounds by high‐performance affinity chromatography; the binding mechanism of the interesting compound to the receptor was investigated by zonal elution and molecular docking. Sinapine in Semen sinapis was proved to be the bioactive compound that specifically binds to the immobilized receptor. The association constant of sinapine to β₂‐AR was determined to be 1.36 × 10⁵ M^?1 with a value of 1.27 × 10^?6 M for the number of binding sites. Ionic bond was believed to be the driving force during the interaction between sinapine and β₂‐AR. It is possible to become a powerful alternative for rapid screening of bioactive compounds from a complex matrix such as traditional Chinese medicine and further investigation on the drug–receptor interaction. Copyright © 2015 John Wiley & Sons, Ltd.     

Hybrid MC/QC simulations of water-assisted proton transfer in nucleosides. Guanosine and its analog acyclovir

To provide an in-depth insight into the molecular basis of spontaneous tautomerism in DNA and RNA base pairs, a hybrid Monte Carlo (MC)–quantum chemical (QC) methodology is implemented to map two-dimensional potential energy surfaces along the reaction coordinates of solvent-assisted proton transfer processes in guanosine and its analog acyclovir in aqueous solution. The solvent effects were simulated by explicit inclusion of water molecules that model the relevant part of the first hydration shell around the solute. The position of these water molecules was estimated by carrying out a classical Metropolis Monte Carlo simulation of dilute water solutions of the guanosine (Gs) and acyclovir (ACV) and subsequently analyzing solute–solvent intermolecular interactions in the statistically-independent MC-generated configurations. The solvent-assisted proton transfer processes were further investigated using two different ab initio MP2 quantum chemical approaches. In the first one, potential energy surfaces of the ‘bare’ finite solute–solvent clusters containing Gs/ACV and four water molecules (MP2/6-31+G(d,p) level) were explored, while within the second approach, these clusters were embedded in ‘bulk’ solvent treated as polarizable continuum (C-PCM/MP2/6-31+G(d,p) level of theory). It was found that in the gas phase and in water solution, the most stable tautomer for guanosine and acyclovir is the 1H-2-amino-6-oxo form followed by the 2-amino-6-(sZ)-hydroxy form. The energy barriers of the water-assisted proton transfer reaction in guanosine and in acyclovir are found to be very similar – 11.74 kcal mol^?1 for guanosine and 11.16 kcal mol^?1 for acyclovir, and the respective rate constants (k = 1.5?×?10¹ s^?1, guanosine and k = 4.09?×?10¹ s^?1, acyclovir), are sufficiently large to generate the 2-amino-6-(sZ)-hydroxy tautomer. The analysis of the reaction profiles in both compounds shows that the proton transfer processes occur through the asynchronous concerted mechanism.     

Thermodynamics of the interaction of daunomycin with DNA

The association constant for the interaction of daunomycin with DNA was determined as a function of temperature (using [³H] daunomycin in conventional equilibrium dialysis cells) and ionic strength (using a spectrophotometric titration method). The association constant varied between 3.1 × 10⁶ M^?1 (4°C) and 3.9 × 10⁵ M^?1 (65°C). The free energy change was ?8.2 to ?8.8 $\text{kcal}\text{mol}$, the enthalpy change ?5.3 $\text{kcal}\text{mol}$ and the entropy change +10 to +11 eu, all values being consistent with that expected of an intercalation process. The apparent number of intercalation sites detected (0.15 to 0.16 per nucleotide) was independent of temperature. The large positive entropy change accompanying the interaction appeals to be due to extensive release of water from the DNA and daunomycin. The apparent number of binding sites increased dramatically with decrease of ionic strength, although the apparent association constant remained largely unaffected by ionic strength.     

Studies on nucleic acids. VIII. Changes in the stability of DNA secondary structure by interaction with divalent metal ions

The melting temperature of a natural DNA is decreased in the presence of increasing amounts of copper ions, whereas other divalent metal ions stabilize the DNA secondary structure at low ionic strength. At 1.28 × 10^?4M, Cu²⁺ produces a decrease of T_m depending on base composition. At very low Cu²⁺ concentrations (0.5 Cu²⁺/2 DNA-P) a stabilization of the DNA conformation appears due to an interaction between Cu²⁺ and phosphate groups of the DNA molecule. In this case the normal trend of GC dependence of T_m exists similar to that with Na⁺ and Mg²⁺ as counterions. If copper ions are in excess, the observed destabilization is stronger for DNAs rich in guanine plus cytosine than for those rich in adenine plus thymine. A sharp decrease of T_m occurs between 0.5–0.8 Cu²⁺/2 DNA-P and 1.5 Cu²⁺/2 DNA-P. The breadth of the transition decreases at high Cu²⁺ concentration with further addition of copper ions. Denaturation and renaturation experiments indicate that Cu²⁺ ions exceeding the phosphate equivalents interact with the bases and reduce the forces of the DNA helix conformation. Evidence is presented, that the destabilization effect produced by Cu²⁺ is possibly due to an interaction with guanine sites of the DNA molecule.     

Theory of transport in linear biological systems: II. Multiflux problems

If in a multiflux system theith flux is given by the integral equation, , the corresponding equation in the Laplace transforms is Γ _i = Σ _j W _ij Γ _j +M _i -the entire system having the matrix formulaion, [I−W]Γ=M. The general solution of this equation and its physical interpretation are discussed. Explicit solutions are given for the general mammillary and catenary systems and for some capillary exchange problems. The theory is applied to the integrated from of the fundamental continuity equation to give equations for total quantity of material in the various “compartments.” If the compartments are uniformly mixed, the integral equation treatment is shown to be mathematically equivalent to the usual differential equation formulation.     

Supportive breeding boosts natural population abundance with minimal negative impacts on fitness of a wild population of Chinook salmon

While supportive breeding programmes strive to minimize negative genetic impacts to populations, case studies have found evidence for reduced fitness of artificially produced individuals when they reproduce in the wild. Pedigrees of two complete generations were tracked with molecular markers to investigate differences in reproductive success (RS) of wild and hatchery‐reared Chinook salmon spawning in the natural environment to address questions regarding the demographic and genetic impacts of supplementation to a natural population. Results show a demographic boost to the population from supplementation. On average, fish taken into the hatchery produced 4.7 times more adult offspring, and 1.3 times more adult grand‐offspring than naturally reproducing fish. Of the wild and hatchery fish that successfully reproduced, we found no significant differences in RS between any comparisons, but hatchery‐reared males typically had lower RS values than wild males. Mean relative reproductive success (RRS) for hatchery F₁ females and males was 1.11 (P = 0.84) and 0.89 (P = 0.56), respectively. RRS of hatchery‐reared fish (H) that mated in the wild with either hatchery or wild‐origin (W) fish was generally equivalent to W × W matings. Mean RRS of H × W and H × H matings was 1.07 (P = 0.92) and 0.94 (P = 0.95), respectively. We conclude that fish chosen for hatchery rearing did not have a detectable negative impact on the fitness of wild fish by mating with them for a single generation. Results suggest that supplementation following similar management practices (e.g. 100% local, wild‐origin brood stock) can successfully boost population size with minimal impacts on the fitness of salmon in the wild.     

Further studies on the specific interaction of S-100 protein with synaptosomal particulates.

Abstract— The specific interaction of S-100 protein with disrupted synaptosomes was further investigated. The specific binding is a saturable and reversible process, and is time, temperature, and strictly Ca²⁺ -dependent. Two affinities affect the interaction (K_ins= 7.04 × 10^?9 M. 1.28 × 10¹² binding sites/ mg protein; K_ins2= 3.91 × 10^?7M, 2.96 × 10¹³ binding sites/mg protein). The half-saturation time is about 5.5 min at 37°C. The half-life of the complex is 17 min at 37°C. At 0°C the binding is 75% slower than at 37° C, and only one-third of the binding sites are involved. The binding capacity is decreased by high NaCl concentrations and by pretreating membranes at high temperatures. Digestion of membranes with trypsin practically abolishes the specific binding. Treatment of membranes with phospholipase C decreases the specific binding, while phospholipase D enhances it to some extent. Other lipid extractors decrease significantly the extent of the interaction. Synaptic plasma membranes seem to be the synaptosomal component involved in the high affinity binding. The S-100 binding activity seems to undergo developmental changes, the adult values of kinetic parameters being reached around the 16th postnatal day in the rat. The results are discussed also in relation to the membrane-bound fraction of S-100.     

Length–weight relationship and seasonal condition in Capoeta sieboldii in the upper Çoruh River,Turkey

Seasonal variations in Fulton’s condition factor (K) and the length–weight relationship (LWR) were investigated for Capoeta sieboldii in the upper Çoruh River (Turkey) from March 2000 to February 2001. There was a slight seasonal variation in condition, with generally lower values in summer and winter, and higher values in autumn. LWR was calculated as W = 0.016 × L^2.933, W = 0.014 ×L^3.001, and W = 0.009 × L^3.118 for males, females, and juveniles, respectively.     

Fetal lung and placental methylation is associated with in utero nicotine exposure

In utero smoke exposure has been shown to have detrimental effects on lung function and to be associated with persistent wheezing and asthma in children. One potential mechanism of IUS effects could be alterations in DNA methylation, which may have life-long implications. The goal of this study was to examine the association between DNA methylation and nicotine exposure in fetal lung and placental tissue in early development; nicotine exposure in this analysis represents a likely surrogate for in-utero smoke. We performed an epigenome-wide analysis of DNA methylation in fetal lung tissue (n = 85, 41 smoke exposed (48%), 44 controls) and the corresponding placental tissue samples (n = 80, 39 smoke exposed (49%), 41 controls) using the Illumina HumanMethylation450 BeadChip array. Differential methylation analyses were conducted to evaluate the variation associated with nicotine exposure. The most significant CpG sites in the fetal lung analysis mapped to the PKP3 (P = 2.94 × 10^?03), ANKRD33B (P = 3.12 × 10^?03), CNTD2 (P = 4.9 × 10^?03) and DPP10 (P = 5.43 × 10^?03) genes. In the placental methylome, the most significant CpG sites mapped to the GTF2H2C and GTF2H2D genes (P = 2.87 × 10^?06 ? 3.48 × 10^?05). One hundred and one unique CpG sites with P-values < 0.05 were concordant between lung and placental tissue analyses. Gene Set Enrichment Analysis demonstrated enrichment of specific disorders, such as asthma and immune disorders. Our findings demonstrate an association between in utero nicotine exposure and variable DNA methylation in fetal lung and placental tissues, suggesting a role for DNA methylation variation in the fetal origins of chronic diseases.     

A new quaternary photoluminescence enhancement system of Eu−N‐(o‐vanillin)‐1,8‐diaminonaphthalene−1,10‐phenanthroline−Zn and its application in determining trace amounts of europium and zinc

A new sensitive quaternary photoluminescence enhancement system has been successfully developed to determine trace amounts of Eu³⁺ and Zn²⁺. The photoluminescence intensity of Eu ? N‐(o‐vanilin)‐1,8‐diaminonaphthalene systems was greatly increased by the addition of specific concentrations of 1, 10‐phenanthroline and Zn²⁺. The excitation and emission wavelengths were 274 and 617 nm, respectively. Under optimal system conditions, the photoluminescence intensity showed a linear response toward Eu³⁺ in the range of 5.0 × 10^–6 ~ 2.0 × 10^–5 M with a limit of detection (= 2.2 × 10^–9 M) and the photoluminescence intensity of the system decreased linearly by increasing the Zn²⁺ concentration in the range of 5.0 × 10^–8 ~ 1.0 × 10^–6 M with a limit of detection (= 8.8 × 10^–11 M). This system was successfully applied for the determination of trace amounts of Eu³⁺ in a high purity La₂O₃ matrix and in the synthetic rare earth oxide mixture, and of Zn²⁺ in a high purity Mg(NO₃)₂ · 6H₂O matrix and in synthetic coexisting ionic matrixes. The energy transfer mechanism, photoluminescence enhancement of the system and interference of other lanthanide ions and common coexisting ions were also studied in detail. Copyright © 2013 John Wiley & Sons, Ltd.     

Application of modified ising model to the helix-coil transition of DNA molecules

Equilibrium properties of heterogeneous DNA near the melting temperature T_m are investigated using the grand partition function. The present approach gives exact and analytical solutions. The algebraic expressions enhance a more thorough understanding of the correlation among many observed equilibrium phenomena. The following quantities have been examined: melting temperature T_m, transition width W, partial melting curves θ_AT and θ_GC, mean length of a helical segment h, and correlation length γ.     

Phosphomannose-isomerase (<Emphasis Type="Italic">pmi</Emphasis>) gene as a selectable marker for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of rapeseed

Nodal segments (4 ± 1 mm long) of Hibiscus moscheutos (hardy hibiscus) were excised from in vitro proliferating microshoots and utilized to evaluate initial factors involved in bulk alginate encapsulation. The factors evaluated were; storage vessel type, volume and multiple rinse effects of CaCl₂ solutions, and sodium alginate concentrations (2.5, 2.75, 3.0 or 3.25%) for bulk alginate encapsulation. Results indicate that vessels utilized for bulk alginate encapsulation should have a lower base area (L × W) to height ratio to reduce the amount of alginate matrix shrinkage resulting in exposure of nodal segments. Increased volumes and multiple 50 mM CaCl₂ solution rinses did not have an effect on alginate solidification. Shoot length, root number, and root length significantly decreased in a linear manner from nodal explants stored for 4 weeks with increasing concentrations of sodium alginate. This research suggests an innovative technique for alginate encapsulation of H. moscheutos utilizing bulk methods as an alternative to single bead alginate encapsulation.     

CELL-WALL FORMATION DURING THE CELL CYCLE OF PORPHYRIDIUM SP. (RHODOPHYTA)

The cell wall of the red microalgae Porphyridium sp. (UTEX 637) comprises a complex amorphous polysaccharide (6–7 × 10⁶ Da). The polysaccharide is made up of xylose, glucose, and galactose as the main sugars, as well as some minor sugars, protein, sulfate, and glucuronic acid, the latter two conferring a negative charge on the polysaccharide. In this study, we used synchronized cultures as one of the ways of unraveling the mechanism of biosynthesis of this complex polysaccharide by following cell-wall formation during the cell cycle. Synchronization of Porphyridium sp. was achieved with an alternating light:dark regime of 12:12 h LD and dilution of the culture at the end of the cycle. Under these conditions, cell duplication occurred between the 12th and 14th hours of the cycle. The following order of building toward formation of the final polysaccharide appeared to take place: Intermediate polysaccharides with molecular masses ranging from 0.5 × 10⁶ to 2 × 10⁶ Da appeared in succession during hours 2–6 of the cycle, and the full-sized polysaccharide was detected by the 8th hour. At the beginning of the cycle, xylose was the predominant sugar. Sulfur peaked at hours 2–4; glucose, galactose, and glucuronic acid at hours 8–12; and the minor sugars at hours 12–14. Upon incubation of low molecular mass polymer (0.5 × 10⁶ Da) collected from the 4th hour with cellular crude extract from cells of the 6th hour of the cycle, two intermediates were formed (0.8 × 10⁶ Da and 2 × 10⁶ Da). We suggest that the 0.5 × 10⁶ Da polymer intermediate, which is composed mainly of xylose, is the first polymer secreted into the medium, where it is further polymerized enzymatically to produce the 2 × 10⁶ Da polymer via an intermediate 0.8 × 10⁶ Da polymer. Later, the full-size polysaccharide is produced.     

Interactions of DNA with divalent metal ions. I. 31P-nmr studies

³¹P-nmr has been used to investigate the specific interaction of three divalent metal ions, Mg²⁺, Mn²⁺, and Co⁺², with the phosphate groups of DNA. Mg²⁺ is found to have no significant effect on any of the ³¹P-nmr parameters (chemical shift, line-width, T₁, T₂, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn²⁺ and Co²⁺, on the other hand, significantly change the ³¹P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn²⁺ and Co²⁺, respectively, corresponding to only 15 ± 5% of the total bound Mn²⁺ and Co²⁺ being directly coordinated to the phosphate groups (inner-sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer-sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the ³¹P relaxation rates of DNA in the absence and presence of Mn²⁺ and Co²⁺ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two-step model was used in the analysis of the kinetic data. The lifetimes of the inner-sphere complexes are 3 × 10^?7 and 1.4 × 10^?5 s for Mn²⁺ and Co²⁺, respectively. The rates of formation of the inner-sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate-determining step in the formation of the inner-sphere complexes. Competition experiments demonstrate that the binding of Mg²⁺ ions is 3–4 times weaker than the binding of either Mn²⁺ or Co²⁺. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (～15%), the higher binding strength of Mn²⁺ and Co²⁺ may be attributed either to base binding or to formation of stronger outer-sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner-sphere phosphate binding increases. In the presence of very high levels of Mg²⁺ (e.g., 3.1M), the inner-sphere ? outer-sphere equilibrium is shifted toward ～100% inner-sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented.     

Retention model for the resolved enantiomers of felodipine on chiral-AGP using micellar mobile phases

A retention model for the chiral separation of an uncharged solute, felodipine, on CHIRAL-AGP, using a micellar mobile phase is proposed. The model assumes the presence of two stereoselective sites and each enantiomer was found to interact with different sites. Addition of a chiral aliphatic alcohol, (+)-(S)-2-octanol, preferentially interacted with the binding site for (?)-(S)-felodipine. The monomeric form of the micellar agent (Tween® 20) competed with the enantiomers for the adsorption sites, and the formation of a 1:1 complex between the enantiomers and the micelles was assumed. The retention of the solutes was effectively controlled by adding small quantities (<1.63 × 10^?3 M) of the nonionic detergent Tween 20 to the mobile phase. Baseline separation was achieved by addition of 1.0 mM n-octylamine to the mobile phase; 8.14 × 10^?4 M Tween 20 in phosphate buffer pH 7.0. The separation factor (α = 1.74) was unaffected by the detergent concentration in the presence of 1.0 mM n-octylamine. © 1995 Wiley-Liss, Inc.     

The non-specific role of Mg2+ in ribosomal subunit association: kinetics and equilibrium in the presence of other divalent metal ions.

The effects of Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺ on the kinetics and equilibrium of the association of vacant “tight” ribosomal subunits from Escherichia coli were studied. Increments of Mg²⁺, Ca²⁺, Sr²⁺ and, by and large, Ba²⁺, to ribosomes dissociated to 30 S and 50 S particles at 1.2 mm-Mg²⁺ (60 mm-M²⁺, pH 7.5, 25°C) produce nearly indistinguishable association curves, with midpoints at 1.8 mm total M²⁺ and complete association to 70 S particles at 4 to 5 mm total M²⁺ . The association rate constants at 1 mm-Mg²⁺, 2 mM-M²⁺ are similar (0.5 × 10⁶ to 0.9 × 10⁶m^?1s^?1), as are the dissociation rate constants at 1 mm-(Mg²⁺ + M²⁺) (0.2 to 0.4 s^?1). Mn²⁺ and Zn²⁺ increase the degree of association, as well as further aggregation (Zn²⁺ especially), at lower concentrations than the alkaline earth ions. Co²⁺ and Ni²⁺ produce lower degrees of association, by promoting dissociation of the 70 S particle : the association rate constants at 1 mm-Mg²⁺, 2 mm-M²⁺ for the transition metal ions are all grouped at 2 × 10⁶ to 3 × 10⁶m^?1s^?1. Ni²⁺ also causes a slower inactivation of one or both subunits.The results are compatible with the view that the effects on the rate and equilibrium constants arise from decreases in the electrostatic free energies of the 30 S, 50 S and 70 S particles produced by large-scale, relatively indiscriminate, charge-neutralization “binding” of M²⁺ , and are difficult if not impossible to reconcile with a specific-sites mode of action of M²⁺.     

Confirming therapeutic target of protopine using immobilized β2‐adrenoceptor coupled with site‐directed molecular docking and the target‐drug interaction by frontal analysis and injection amount–dependent method

Drug‐protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β₂‐adrenoceptor (β ₂‐ AR) by linkage of the receptor on macroporous silica gel surface through N ,N ′‐carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site‐directed molecular docking. Subsequent application of immobilized β ₂‐ AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount–dependent method. The association constants of protopine to β ₂‐ AR by the 2 methods were (1.00 ± 0.06) × 10⁵M⁻¹ and (1.52 ± 0.14) × 10⁴M⁻¹. The numbers of binding sites were (1.23 ± 0.07) × 10⁻⁷M and (9.09 ± 0.06) × 10⁻⁷M, respectively. These results indicated that β ₂‐ AR is the specific target for therapeutic action of protopine in vivo. The target‐drug binding occurred on Ser¹⁶⁹ in crystal structure of the receptor. Compared with frontal analysis, injection amount–dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high‐throughput drug‐receptor interaction analysis.