The Influence of pH on the Cadmium-Repressed Growth of the Alga Coelastrum proboscideum

The inhibition of growth by different concentrations of CdCl₂ in the range 4,5 × 10^?7 to 5.6 × 10^?7M was studied in the green alga Coelastrum proboscideum Bohlin in inorganic media at pH 4.3, 5.3 and 6.2. The factorial destgn of the experiments was evaluated as an analysis of 2² factors. Below pH 4.0 and above pH 6.5 growth was depressed without adding Cd. Cd concentrations exceeding 5.6 × 10^?8M reduced algal growth significantly with a 50% inhibition at 5.6 × 10^?7M Cd. The Cd concentration of 5.6 × 10^?7M was less toxic at pH 6.2 than at pH 5.3 and 4.3, thus revealing a negative interaction between protons and Cd.     

Specific thyroxine receptors in mammary cytosol from lactating cattle

Specific thyroxine (T₄) binding was identified in bovine mammary cytosol preparations. Binding specificity of 3,5,3′ triiodothyronine (T₃) with respect to T₄ was less than 1%. The halftime of T₄ binding and displacement at 4°C was 1 and 20 minutes, respectively. Scatchard analyses demonstrated the presence of two T₄ binding components with dissociation constants of 3.61 × 10^?10 and 7.73 × 10^?8M. The high affinity binding component had a molecular weight of ～ 100,000, a T₄ binding capacity of 5.05 × 10^?12moles/mg protein, and was destroyed by boiling or protease treatment. High-affinity, low capacity T₄ binding was not found in bovine serum and was unique to mammary tissue.     

Chymotrypsin inhibitor from potatoes: interaction with target enzymes

The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The K_i value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10^?10M, while the second-order rate constant for association was 6 × 10⁵ M^?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a K_i value of 9.6 × 10^?9 M. The inhibitor also exhibited a tight association with subtilisin (K_i < 4 × 10^?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH.     

In vitro triiodothyronine binding to non-histone proteins from rat liver nuclei

$\text{In vitro}$ incubations of non-histone proteins from rat liver nuclei with labelled L-3, 5, 3′ triiodothyronine demonstrate the existence of high affinity, limited capacity binding sites for the hormone in this protein group; the affinity was found identical for triiodothyroacetic acid and lower for L-thyroxine. Binding ability was highly temperature dependent. At 4°C, the rate constant of association was 0.9 × 10⁷ M^?1 h^?1 and the rate constant of dissociation was 0.015 h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 1.6 and 5 × 10^?9 M. The maximum binding capacity was 10^?13 moles of L-3, 5, 3′ triiodothyronine per 100 μg non-histone proteins or 6000 hormone molecules per nucleus. Protein binding had a half-life of 20 hours at 4°C, in the absence of hormone, but was found to be very stable in the presence of hormone.     

Understanding nucleosomal histone and DNA interactions: a biophysical study

Histones are associated with DNA to form nucleosome essential for chromatin structure and major nuclear processes like gene regulation and expression. Histones consist of H1, H2A, H2B and H3, H4 type proteins. In the present study, combined histones from calf thymus were complexed with ct DNA and their binding affinities were measured fluorimetrically. All the five histones were resolved on SDS page and their binding with DNA was visualized. The values of biding affinities varied with pH and salt concentration. Highest affinity (4.0?×?10⁵ M^?1) was recorded at pH 6.5 in 50 mM phosphate buffer and 1.5?×?10⁴ M^?1 in 2 M NaCl at pH 7.0. The CD spectra support the highest binding affinity with maximum conformational changes at pH 7.0. The time-resolved fluorescence data recorded two life times for histone tyrosine residues at 300 nm emission in phosphate buffer pH 6.5. These life times did not show much change upon binding with DNA in buffer as well as in 2 M NaCl. The isothermal calorimetric studies yielded thermodynamic parameters ΔG, ΔH and ΔS as ?1.6?×?10⁵ cal/mol, ?1.13?×?10³ cal/mol and ?3.80 cal/mol/deg, respectively, evidencing a spontaneous exothermic reaction. The dominant binding forces in building the nucleosome are electrostatic interactions.     

Studies on Nicotinoids as an Insecticide

In search for the mechanism of insecticidal action of nicotinoids, the kinetics of house fly head cholinesterase inhibition by nicotine were studied to determine the type of inhibition. The pH dependency of inhibition was interpreted in terms of protonation of nitrogen atom in the molecule and the inhibition was shown to be the mixed type closing to competitive type. The Michaelis constants are 3.5 × 10^?4 M and 4.1 × 10^?4 M, while the apparent inhibition constants obtained are 1.0 × 10^?3 M and 2.3 × 10^?3 M at pH 7.4 and 8.4, respectively. The type of the inhibition by nicotine monomethiodide carring univalent cation was competitive and the apparent inhibition constant is 1.5 × 10^?4 M. These data indicated that the cationic head of nicotinium ion interacts with the anionic site in the active center of cholinesterase.     

In vitro triiodothyronine binding to cytoplasmic proteins from human red blood cells.

In vitro incubations of cytosol proteins from human red blood cells with [¹²⁵I] labelled L-3,5,3′ triiodothyronine demonstrated the existence of high affinity and limited capacity binding sites for T₃. At 4°C, the rate constant of association was 3 × 10⁷ M^?1h^?1, and the rate constant of dissociation was 9.10^?3h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 3 and 7.10^?10M. The maximum binding capacity was 1.4 f moles of L-3,5,3′ triiodothyronine per mg cytosol proteins. A close parallel between the biological pontency of the analogs of L-T₃ was observed.     

Kinetics of cyanide binding to chloroperoxidase in the presence of nitrate: detection of the influence of a heme-linked acid group by shift in the appa

The kinetics of the binding of cyanide to ferric chloroperoxidase have been studied at 25°C and ionic strength 0.11 M using a stopped-flow apparatus. The dissociation constant (K_CN) of the peroxidase-cyanide complex and both forward (k₊) and reverse (k_?) rate constants are independent of the H⁺ concentration over the pH range 2.7 to 7.1. The values obtained are k_cn = (9.5 ± 1.0) × 10^-5 M, k₊. = (5.2 ± 0.5) × 10⁴ M^?1 sec^?1 and k_- = (5.0± 1.4) sec^-1. In the presence of 0 06 M potassium nitrate the affinity of cyanide for chloroperoxidase decreases due to the inhibition of the forward reaction. The dissociation rate is not affected. The nitrate anion exerts its influence by binding to a protonated form of the enzyme, whereas the cyanide binds to the unprotonated form. Binding of nitrate results in an apparent shift towards higher pK_a values of the ionization of a crucial heme-linked acid group. Hence the influence of this group can be detected in the accessible pH range. Extrapolation to zero nitrate concentration yields a value of 3.1±0.3 for the pK_a of the heme-linked acid group.     

Kinetics of the stacking of ethidium bromide by the raman laser temperature-jump method

Raman laser temperature-jump measurements have been made on concentrated solutions of ethidium bromide. Two relaxations were observed. The faster has a lifetime of less than 30 ns and is attributed to rotation of the phenyl ring. The slower relaxation is concentration dependent and is due to the parallel stacking of two dye molecules. The forward and reverse rates for this process are (4.6 ± 1.4) × 10⁸ M^?1s^?1 and (6.7 ± 1.4)× 10⁶ s^?1, respectively, at 25°C. 0.25 M ionic strength, and pH 6.9. This reverse rate and those of three similar reactions are found to fit a linear free energy plot. The implications of these results for studies of nucleic acid base stacking are discussed.     

Equilibrium studies on neutral red-DNA binding.

Spectrophotometric binding studies were undertaken on the interaction of neutral red with native and heat-denatured, sonicated, calf thymus DNA in a 0.2M ionic strength buffer containing Tris–sodium acetate–potassium chloride at 25°C. The pK_A of neutral red was found to be 6.81. At pH 5 the binding of protonated neutral red was complicated even at low concentration ratios of dye to DNA. In the pH range 7.5–8.5 the tight binding process could be studied and it was found that both protonated and free base species of neutral red significantly bind with DNA having association constants (in terms of polynucleotide phosphate) of 5.99 × 10³ M^?1 and 0.136 × 10³ M^?1, respectively, for native DNA and 7.48 × 10³ M^?1 and 0.938 × 10³ M^?1, respectively, for denatured DNA. The pK_A value of the neutral red–DNA complexes were 8.46 for native DNA and 7.72 for denatured DNA. These results are discussed in terms of possible binding mechanisms.     

Phosphofructokinase of Solanum tuberosum tuber

Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. K_m for fructose-6-phosphate, ATP and Mg²⁺ was 2·1 × 10^?4 M, 4·5 × 10^?5 M and 4·0 × 10^?4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.     

Specific 3,3′,5′-Triiodothyronine (Reverse T3) Binding Sites on Rat Liver Plasma Membranes: Comparison with Thyroxine (T4) Binding Sites

Abstract

The binding characteristics of thyroxine (T4), triiodothyronine (T3), and reverse T3 (rT3) to rat liver plasma membranes (RLPM) were examined to explore the interactions of thyroid hormones with cell surface receptors. Scatchard analysis suggested that all three ligands bound to two classes of binding sites. The high affinity rT3 binding sites appeared to be distinct from the high affinity T4 sites, on the basis of differing optimum physicochemical conditions for binding, and analog displacement studies. The higher affinity constant for T4 was 1.7 ± 0.2 × 10⁹ M^-1 (mean ± SEM) and binding capacity was 3.1 ± 0.3 pmol mg ^-1 protein whereas for rT3 binding the Ka was 2.5 ± 0.4 × 10⁸ M^-1 and capacity was 6.2 ± 0.9 pmol mg ^-1. (¹²⁵ I) T3 bound with lower affinity and T3 tracer was readily displaced by T4. Moreover, comparatively higher concentrations of T3 were needed to displace either radiolabeled T4 or rT3, suggesting that T3 was binding to both the T4 and rT3 sites with lower affinity. Marker enzyme studies on RLPM, of varying purity prepared by different methods, showed a positive correlation between the activity of the plasma membrane enzyme magnesium-stimulated ATPase and high affinity rT3 and T4 binding. Column chromatography of the radioligands, after dissociation from membrane binding sites, confirmed that the integrity of the hormones was not altered during association or dissociation. Our results raise the possibility that the high affinity T4 and rT3 binding sites on RLPM may be hormone receptors mediating biological actions at the membrane level.     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. K_I, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k₃, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. K_I values of 1.3 × 10^{? 4} M^{? 1}, 5.6 × 10^{? 6} M^{? 1} and 7.2 × 10^{? 6} M^{? 1} were obtained for malathion, malaoxon and isomalathion, respectively. The IC₅₀ values for free/immobilized AChE, (3.7 ± 0.2) × 10^{? 4} M/(1.6 ± 0.1) × 10^{? 4}, (2.4 ± 0.3) × 10^{? 6}/(3.4 ± 0.1) × 10^{? 6} M and (3.2 ± 0.3) × 10^{? 6} M/(2.7 ± 0.2) × 10^{? 6} M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10 mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 × 10^{? 7} M/2 × 10^{? 7} M, 2 × 10^{? 7} M/3 × 10^{? 7} M and 2 × 10^{? 7} M/4.5 × 10^{? 7} M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.     

The binding of Ni2+ to adenylyl-3',5'-adenosine and to poly(adenylic acid)

Studies of the binding of Ni²⁺ to adenylyl-3',5'-adenosine (ApA) at pH 6-0 by ultraviolet spectrophotometry indicate the formation of a 1:1 complex in the presence of a large excess of metal ion. At 25 °C. and ionic strength μ = 0.5 M, the stability constant of Ni(ApA) is evaluated to be K = 2.6 (±0.6) M^?1. The low stability is taken as evidence that the predominant complex species is one in which the ApA acts as a monodentate ligand, mainly through the adenine group. The rate constants for complex formation and dissociation, k_f = 1430 M^?1 s^?1 and k_b = 665 s^?1 (25°C. μ = 0.5M). determined by the temperature-jump relaxation technique, are consistent with this interpretation. The binding strength of Ni²⁺ to poly(adenylic acid) [poly(A)] has been studied at pH 7.0 using murexide as an indicator of the concentration of free Ni²⁺. Within the concentration range [Ni²⁺ = 1 × 10^?5 × 10^?3 M the data can be represented in the form of a linear Scatchard plot. i.e., the process can be described as the binding of Ni²⁺ to one class of independent binding sites. The number of binding sites per monomer is 0.26, and the stability constant K = 8.2×10³ M^?1 (25°C μ = 0.1 M). In kinetic studies of the reaction of Ni²⁺ with poly(A), two relaxation effects due to complex formation were detected, one with a concentration-independent time constant of about 0.4 ms, the other with a concentration-dependent time constant in the millisecond range. The concentration dependence of the longer relaxation time can be accounted for by a three-step mechanism which consists of a fast second-order association reaction followed by two first-order steps. There is evidence, however, that the overall process is more complicated than expressed by the three-step mechanism.     

Characterization and Regulatory Properties of Glucose-6-Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The K_m value for glucose-6-phosphate is 1.6 × 10^?4 and 6.3 × 10^?4M at low (1.0–6.0 × 10^?4M) and high (6.0–30.0 × 10^?4M) concentrations of the substrate, respectively. The K_m value for NADP⁺ is 1.4 × 10^?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg²⁺, and Mn²⁺. In the presence of NADPH, the plot of activity vs. NADP⁺ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg²⁺ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.     

In vitro stimulation of human red blood cell Ca2+-ATPase by thyroid hormone

Ca²⁺-ATPase activity in human erythrocyte ghosts previously washed to remove endogenous thyroid hormone is stimulated $\text{in}\text{vitro}$ by physiologic concentrations of thyroxine (T₄) and triiodothyronine (T₃). Two- to three-fold increases (P <0.005) in Ca²⁺-ATPase activity occurred after 60–120 minutes' exposure of membranes to iodothyronines at concentrations of T₄ and T₃ of 10^?8 M to 10^?12 M. T₄ was more active than T₃ and its activity did not depend upon prior conversion to T₃. The Ca²⁺-ATPase effect represents an extranuclear action of thyroid hormone in a human cell model.     

Effect of guanyl nucleotides on the stimulation of adenyl cyclase activity in human thyroid plasma membranes by thyroid-stimulating hormone and prostaglandin E2

Thyroid homogenates and thyroid plasma membranes were prepared from human thyroid and the effects of thyroid-stimulating hormone (thyrotropin), NaF, and prostaglandins E₁ and E₂ on adenyl cyclase activity in these preparations were studied. The basal level of adenyl cyclase activity in plasma membranes was 5–8 times greater than that of the original homogenates. Adenyl cyclase activity in plasma membranes was stimulated 4.7-fold by 100 munits/ml of thyrotropin and 5-fold by 10 mM of NaF, but the activity in the homogenates was only stimulated 2-fold by either thyrotropin or NaF. Prostaglandin E₁ (10^?6?10^?3 M) and prostaglandin E₂ (10^?7?10^?4 M) failed to stimulate adenyl cyclase activity in plasma membranes, but they did stimulate adenyl cyclase activity in the homogenates. A marked stimulatory effect of prostaglandin E₂ (10^?5 M) on adenyl cyclase activity in plasma membranes resumed in the presence of GTP (10^?7?10^?4 M), although GTP itself only slightly stimulated enzyme activity. GDP and GMP were also effective in this respect, although their potencies varied from compound to compound. GTP potentiated slightly the action of thyrotropin on adenyl cyclase in plasma membranes, but it significantly depressed an increase of enzyme activity produced by NaF. Since GTP did not affect the ATP-regenerating system, it seems that GTP, GDP or GMP was required for the manifestation of prostaglandin E₂ action on adenyl cyclases of human thyroid plasma membranes.     

Thermodynamic and kinetic parameters of ion condensation to polynucleotides: Outer sphere complex formed by Mg++ ions

The coupling of ion binding to the single strand helix—coil transition in poly (A) and poly(C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly(C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M^?1 for the ion binding to the polymer. The rate constant of helix formation is found to be 1.3 × 10⁷ s^?1, whereas the dissociation rate is 1.0 × 10⁶ s^?1. Similar data are presented for poly (A) and poly (dA).The interaction of Mg⁺⁺ and Ca⁺⁺ with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding “sites”. The stability constants increase with decreasing salt concentration from 2.7 × 10⁴ M^?1 at medium ionic strengths up to 2.7 × 10⁷ M^?1 at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg⁺⁺ binding are characterised over a broad range of Mg⁺⁺ concentrations from 5 μM to 500 μM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg⁺⁺ and Ca⁺⁺ association to the polymer is close to the limit of diffusion control (k_R = 1 × 10¹⁰ to 2 × 10¹⁰ M^?1 s^?1). This high rate demonstrates that Mg⁺⁺ and Ca⁺⁺ ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg⁺⁺ or Ca⁺⁺, and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg⁺⁺ and Ca⁺⁺ surround the polynucleotides in the form of a mobile ion cloud without site binding.