Phosphorylation of cottonplant proteins under the action of protein kinase inhibitors and activators

The occurrence of Ca²⁺-, Ca²⁺-phospholipid-, Ca²⁺-calmodulin-, and cAMP-dependent phosphorylation has been shown with the aid of protein kinase activators and inhibitors and by electrophoresis and autoradiography. Specific substrates have been revealed and it has been demonstrated that the pathways of the realization of the action of cAMP-dependent and Ca²⁺-calmodulin-dependent protein kinases may intersect.A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax 627071. Institute of Nuclear Physics, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax 442603. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 96–100, January–February, 1994.     

Comparison of Ca2+ and Mg2+ binding to calmodulin. An enthalpy,entropy, and gibbs free energy analysis

A consistent set of G _B , H _B , and S _B parameters have been determined from ion specific electrode, calorimetric, and spectrophotometric studies for the binding of Ca²⁺ and Mg²⁺ to bovine calmodulin at pH=7.0 and an ionic strength I of 0.113M. A non-linear least squares analysis of calcium specific ion electrode data yields, on a molar basis, four calcium dissociation constants: 10^–7 for the first site, 10^–5 for the fourth site, and two constants between these values. Both calorimetric experiments and an indicator method provide evidence that Mg²⁺ binds to calmodulin, probably at the same sites as Ca²⁺, but with affinities about 100 times smaller: 4×10^–5 for the first site and 2×10^–3 for the fourth. Calorimetric titrations on Ca²⁺ binding to calmodulin in three buffers are consistent with 0.46 protons released upon binding at all four sites and yield an average H _B per site of 5.6 and 7.9 kJ-mol^–1 for Ca²⁺ and Mg²⁺, respectively. The entropy of the system increases by 524 and 361 J-K^–1-mol^–1 when Ca²⁺ and Mg²⁺, respectively, bind to four sites on calmodulin, i.e., the selectivity of calmodulin for Ca²⁺ is primarily derived from entropy effects. Further analysis based on elimination of the entropy term for the metal ions demonstrates that calmodulin bound to Ca²⁺ has a larger entropy than the unbound calmodulin; the opposite is true for calmodulin bound to Mg²⁺. These analyses are consistent with the hypothesis that Ca²⁺ forms tight complexes at all sites on calmodulin and that release of waters of hydration upon binding is the source of the increase of entropy in the system.     

Inhibition of Glutamate Release from Rat Cortical Nerve Terminals by Dehydrocorydaline,an Alkaloid from Corydalis yanhusuo

Excessive release of glutamate induces excitotoxicity and causes neuronal damage in several neurodegenerative diseases. Natural products have emerged as potential neuroprotective agents for preventing and treating neurological disorders. Dehydrocorydaline (DHC), an active alkaloid compound isolated from Corydalis yanhusuo, possesses neuroprotective capacity. The present study investigated the effect of DHC on glutamate release using a rat brain cortical synaptosome model. Our results indicate that DHC inhibited 4-aminopyridine (4-AP)-evoked glutamate release and elevated intrasynaptosomal calcium levels. The inhibitory effect of DHC on 4-AP-evoked glutamate release was prevented in the presence of the vesicular transporter inhibitor bafilomycin A1 and the N- and P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC but not the intracellular inhibitor of Ca²⁺ release dantrolene or the mitochondrial Na⁺/Ca²⁺ exchanger inhibitor {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157. Moreover, the inhibitory effect of DHC on evoked glutamate release was prevented by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) inhibitor PD98059. Western blotting data in synaptosomes also showed that DHC significantly decreased the level of ERK1/2 phosphorylation and synaptic vesicle-associated protein synapsin I, the main presynaptic target of ERK. Together, these results suggest that DHC inhibits presynaptic glutamate release from cerebrocortical synaptosomes by suppressing presynaptic voltage-dependent Ca²⁺ entry and the MAPK/ERK/synapsin I signaling pathway.     

Protein kinase from cotton seeds

A Ca²⁺-dependent protein kinase C, active at pH 6.5–8.0 has been found in cotton seeds for the first time. The localization of the enzyme in the seeds has been established and some of its properties are described (stability in various media, capacity for performing the phosphorylation of various substrates, activation by calcium ions). Highly active preparations of cottonseed protein kinase C have been isolated by biospecific chromatography.Institute of Bioorganic Chemistry, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 255–259, March–April, 1989.     

The solubility of CaMoO4(c) and an aqueous thermodynamic model for Ca2+-MoO 4 2 -ion-interactions

The solubility of powellite [CaMoO₄(c)] was studied in aqueous Na₂MoO₄, CaCl₂ and Ca(NO₃)₂ solutions ranging in concentrations from 1×10^–4M to 1.0M and over equilibration times extending to 36 days. Our experimental data were interpreted using the aqueous ion-interaction model of Pitzer and coworkers. The Ca²⁺–MoO ₄ ^2– ion-interactions were found to be analogous to Ca²⁺–SO ₄ ^2– . The use of Ca²⁺–MoO ₄ ^2– ion-interactions parameters (⁽⁰⁾=0.2, ⁽¹⁾ = 3.1973 and ⁽²⁾) and a logK _sp of –7.93 gave excellent predictions of all of the experimental data. Commonion ternary interaction parameters such as MoO ₄ ^2– –Cl^– or MoO ₄ ^2– –NO ₃ ^– were not required.     

An aqueous thermodynamic model for Ca2+−SO 3 2− ion interactions and the solubility product of crystalline CaSO3·1/2H2O

The solubility of CaSO₃·1/2H₂O(c) was studied under alkaline conditions (pH>8.2), in deaerated and deoxygenated Na₂SO₃ solutions ranging in concentration from 0.0002 to 0.4M and in CaCl₂ solutions ranging in concentration from 0.0002 to 0.01M, for equilibration periods ranging from 1 to 7 days. Equilibrium was approached from both the over- and the under-saturation directions. In all cases, equilibrium was reached in <1 days. The aqueous Ca²⁺–SO ₃ ^2– ion interactions can be satisfactorily modeled using either ion-association or ion-interaction aqueous thermodynamic models. In the ion-association model, the log K°=2.62±0.07 for Ca²⁺+SO ₃ ^2– CaSO ₃ ⁰ . In the Pitzer ion-interaction model, the binary parameters (⁰) and (¹) for Ca²⁺–SO ₄ ^2– were used, and the value of (²) was determined from the experimental data. As expected given the strong association constant, the value of (⁰) was quite small (about –134). We feel a combination of the two models is most useful. The logarithm of the thermodynamic equilibrium constant (K°) of the CaSO₃·1/2H₂O(c) solubility reaction (CaSO₃·1/2H₂O(c)Ca²⁺+SO ₃ ²⁺ +0.5H₂O) was found to be –6.64±0.07.     

Quantum-chemical investigation of the localization and hydration of Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> cations in eight- and ten-fold structural rings in the clinoptilolite structure

The hydration of Ca²⁺ and Mg²⁺ exchange cations in solution and in 10- and 8-membered silicon—oxygen rings of the clinoptilolite was studied by ab initio and MNDO quantum-chemical methods. The coordination numbers of these cations with respect to water molecules and their hydration energies were determined. It is shown that the localization of Ca²⁺ and Mg²⁺ in the clinoptilolite structure was different for the dehydrated state and the partially hydrated state. The ion exchange sorption energy calculated for the Ca²⁺—Mg-Cli system was in satisfactory agreement with the experimental data.Translated from Teoreticheskaya i Éksperimentalnaya Khimiya, Vol. 40, No. 6, pp. 357–362, November–December, 2004.     

Twinned and Disordered Crystal Structure of Solvated Bis[Aqua(2.2.2-Cryptand)Calcium] Hexa(Isothiocyanato)Calcium

Twinned and disordered crystals of solvated bis[aqua(2.2.2-cryptand)calcium] hexa(isothiocyanato)calcium 2[Ca(2.2.2-Crypt)(H₂O)]²⁺ · [Ca(NCS)₆]^4– · Sol (I), where Sol is acetone and/or ethanol and may be water, were synthesized and studied by X-ray diffraction analysis. Structure I (space group P2₁/n, a = 11.841 Å, b = 21.787 Å, c = 12.377 Å, = 90.90°) was solved by the direct method and refined by the full-matrix least squares method in anisotropic approximation to R = 0.079 from 4168 independent reflections (CAD4 automated diffractometer, MoK ). In crystal form, complex I exists as the two aforesaid complex ions [I¹]²⁺ and [I²]^4– in the molar ratio 2 : 1 united through hydrogen bonds. Complex cation I¹ is of the guest–host type. Its Ca²⁺ cation is coordinated by all eight heteroatoms (6O + 2N) of the cryptand ligand and by the O atom of the water molecule; the coordination polyhedron of this Ca²⁺ cation (CN 9) is irregular. The Ca²⁺ cation of complex anion I² (in the crystallographic center of inversion) is coordinated by six N atoms of six neighboring SCN^– anionic ligands; the coordination polyhedron of this Ca²⁺ cation (CN 6) is a slightly distorted octahedron.     

Properties of trypsin immobilized on the surface of organosilicas

Trypsin is immobilized on silochromes with polymaleic anhydride (MA-silochrome) or cyanuric chloride (CC-silochrome) grafted to the surface. The differences in the surface charge of these carriers causes differences in the pH-relationships of the rate of hydrolysis of N-benzoyl-D,L-arginine-p-nitroanilide by the immobilized preparations. A considerable increase ( threefold) in the activity of trypsin immobilized on MA-silochrome has been observed, when 10^–2 mole/liter of CaCl₂ was introduced into the solution of the substrate. The activation of the enzyme is reversible and the rate of the catalyzed reaction decreases to the initial rate, when the Ca²⁺ ions are removed by means of EDTA. The enzyme bound to CC-silochrome does not reveal any such effect. The inactivation kinetics of trypsin immobilized on MA-aerosil is complex in character and, in contrast to the native enzyme, is not described by a first-order reaction equation. The immobilized preparation is more stable than the native enzyme. The above-noted effects are probably caused by changes in the properties of the biocatalyst molecule on binding with a solid matrix.Translated from Teoreticheskaya i Éskperimental'naya Khimiya, Vol. 25, No. 1, pp. 113–116, January–February, 1989.     

Interlaboratory exercises to compare analytical method performances

Three intercomparison exercises on simulated rainwater were held in the period 1991–93 involving 72 to 99 laboratories in Europe and South America. The exercises required the analysis of pH, conductivity, main anions (Cl^–, NO ₃ ^– and SO ₄ ^2– ) and main cations (Ca²⁺, K⁺, Mg²⁺, Na⁺ and NH ₄ ⁺ ). The concentrations of the single ions ranged between 5 and 150 moll^–1. Results were used to evaluate and compare the precision of the analytical methods. A general improvement in precision was observed in the course of the exercises.     

Production,Purification and Characterisation of a Potential Fibrinolytic Protease from Endophytic <Emphasis Type="Italic">Xylaria curta</Emphasis> by Solid Substrate Fermentation

The present investigation highlights the optimal conditions for production of a non-toxic, bi-functional fibrinolytic enzyme xylarinase produced by endophytic fungus Xylaria curta by solid substrate fermentation using rice chaff medium. The purified enzyme is a monomeric protein with a molecular mass of ～33 kDa. The enzyme exhibits cleavage of Aα and Bβ chains of fibrin(ogen) and has no effect on γ chain. The optimal fibrinolytic activity of the enzyme was observed at 35 °C and pH 8. The fibrinolytic activity was enhanced in the presence of Ca²⁺, whereas it was completely inhibited in the presence of Fe²⁺ and Zn²⁺ ions and inhibitors like EDTA and EGTA suggesting it to be a metalloprotease. The K _m and V _max of the enzyme for azocasein were 326 μM and 0.13 μM min^?1. The N-terminal sequence of the enzyme (SNGPLPGGVVWAG) was same when compared to xylarinase isolated from culture broth of X. curta. Thus, xylarinase could be exploited as a potent clot busting enzyme which could be produced on large scale using solid substrate fermentation.     

Transglutaminase 2 inhibits apoptosis induced by calciumoverload through down-regulation of Bax

An abrupt increase of intracellular Ca²⁺ is observed in cells under hypoxic or oxidatively stressed conditions. The dysregulated increase of cytosolic Ca²⁺ triggers apoptotic cell death through mitochondrial swelling and activation of Ca²⁺-dependent enzymes. Transglutaminase 2 (TG2) is a Ca²⁺-dependent enzyme that catalyzes transamidation reaction producing cross-linked and polyaminated proteins. TG2 activity is known to be involved in the apoptotic process. However, the pro-apoptotic role of TG2 is still controversial. In this study, we investigate the role of TG2 in apoptosis induced by Ca²⁺-overload. Overexpression of TG2 inhibited the {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-induced apoptosis through suppression of caspase-3 and -9 activities, cytochrome c release into cytosol, and mitochondria membrane depolarization. Conversely, down-regulation of TG2 caused the increases of cell death, caspase-3 activity and cytochrome c in cytosol in response to Ca²⁺-overload. Western blot analysis of Bcl-2 family proteins showed that TG2 reduced the expression level of Bax protein. Moreover, overexpression of Bax abrogated the anti-apoptotic effect of TG2, indicating that TG2-mediated suppression of Bax is responsible for inhibiting cell death under Ca²⁺-overloaded conditions. Our findings revealed a novel anti-apoptotic pathway involving TG2, and suggested the induction of TG2 as a novel strategy for promoting cell survival in diseases such as ischemia and neurodegeneration.     

Modelling of α-lactalbumin from the known structure of hen egg white lysozyme using molecular dynamics

Summary The prediction of protein conformation by homology is being widely pursued using interactive computer graphics. However, there have been a limited number of energy minimization and/or molecular dynamics studies for such predictions. This paper reports one such study on -lactalbumin, a system that can be tested as the X-ray crystal structure has recently been determined. The differences in structure of the Ca²⁺ binding loop reported for the holo-protein (Stuart et al., Nature, 324 [1986] 84–87) and that predicted for the apoprotein could be attributed to the presence or absence of the Ca²⁺ ion.     

Phthaleinviolett als Indicator in der Chelatometrie

Zusammenfassung Durch Dünnschicht-Chromatographie auf Maisstärke konnte die Trennung und der Nachweis von Pb²⁺-, Ag⁺- und Hg₂ ²⁺- sowie Ba²⁺-, Sr²⁺-, Ca²⁺- und Mg²⁺-Ionen durchgeführt werden. Als Fließmittel diente Aceton-3 n Salpetersäure (11) bzw. Aceton-3 n Salzsäure (23). 1 · 10^–8 bis 5 · 10^–9 Äquivalente der Ionen konnten nachgewiesen werden.

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Summary Separation and identification of Pb²⁺, Ag⁺, Hg²⁺ and Ba²⁺, Sr²⁺, Ca²⁺, Mg²⁺ ions has been achieved by thin-layer chromatography on maize starch, using acetone — 3 n nitric acid (11) resp. acetone — 3 n hydrochloric acid (23) as a solvent. 1 · 10^–8 to 5 · 10^–9 equivalents of the ions could be detected.

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Ein Teil der Versuche für diese Arbeit hat die Chemie-Absolventin Ana Bem ausgeführt, wofür ihr auch bei dieser Gelegenheit gedankt sei.     

Complexation properties of phosphonocarboxylic acids in aqueous solutions

The concentration formation constants of phosphonoacetic acid (PAA) complexes with the Ca²⁺ and Mg²⁺ ions were determined in aqueous solution at 25°C by potentiometric and coulometric titrations at different ionic strengths and were extrapolated to I=0 in order to obtain thermodynamic values of the formation constants. Complexes were formed by the completely deprotonated K _f (ML) and monoprotonated K _f (MHL) forms of the PAA anion. The respective values for the complexes are: log K _f (CaL)=4.68±0.03, log K _f (CaHL)=2.61±0.08; log K _f (MgL)=5.58±0.09, log K _f (MgHL)=3.0±0.3. The enthalpy and entropy of complexation for the deprotonated Ca²⁺ and Mg²⁺ PAA species, determined from the temperature dependence of the log K _f (ML), are: H⁰(Ca) =0.6±0.2 kcal-mol^–1, S⁰(Ca)=21.4±0.6 cal-mol^–1-K^–1, H⁰(Mg)=3.0±0.7 kcal-mol^–1, and S⁰(Mg)=35±2 cal-mol^–1-K^–1. It is seen there-fore, that the complexes are entropy stabilized but enthalpy destabilized. Formation constants were also determined for Ca²⁺ and Mg²⁺ complexes with PAA analogs, phosphonoformic and 3-phosphonopropionic acids and the complexation of PAA was also studied at a single ionic strength, with Na⁺, Ag⁺, Tl⁺, Sr²⁺, Ba²⁺, Cd²⁺, Cu²⁺, and Pb²⁺ ions.     

Distribution of Strontium in the System Water — Ca(NO3)2 — Nitrobenzene — Strontium Dicarbollylcobaltate — 18-crown-6

From several strontium distribution experiments with ⁸⁵Sr tracer, the extraction constant corresponding to the equilibrium Ca²⁺(aq)+SrL²⁺(nb) CaL²⁺(nb)+Sr²⁺ (aq) in the two-phase water-nitrobenzene system (L = 18-crown-6; aq = aqueous phase, nb = nitrobenzene phase) was tentatively evaluated as log K _ex (Ca²⁺,SrL²⁺) = –1.9±0.1. Furthermore, the stability constant of the calcium — 18-crown-6 complex in nitrobenzene saturated with water was calculated for a temperature of 25 °C: log _nb(Cal²⁺) = 10.1±0.1.     

Solubility of Crystalline Calcium Isosaccharinate

The solubility of calcium isosaccharinate Ca(ISA)₂(c) was determined at 23°C as a function of pH (1–14) and calcium ion molality (0.03–0.52). The similarity of solubility from the over- and undersaturation directions for different equilibration periods indicated that equilibrium in these solutions was reached rapidly (< 7 days) and that these data can be used to develop thermodynamic equilibrium constants. The solubility data were interpreted using the Pitzer ion–interaction model. The logarithms of the thermodynamic equilibrium constants determined from these data were 1.30 for the dominant reaction at pH < 4.5 [Ca(ISA)₂(c) + 2H⁺ Ca²⁺ + 2HISA(aq)], and –2.22 for the dominant reaction at 4.5 [Ca(ISA)₂(c)+ Ca(ISA)₂(aq)]. In addition, the logarithm of the dissociation constant of HISA [HISA(aq) ISA- + H⁺] was calculated to be –4.46.     

Synthesis and Application of a Novel Poly(Acryl-p-Toluenesulfonamideamidine-p-Toluenesulfonylamide) Chelating Fiber for Preconcentration and Separation of Trace Noble Metal Ions from Solution Samples

A novel poly(acryl-p-toluenesulfonamideamidine-p-toluenesulfonylamide) chelating fiber containing S, N and O elements was synthesized from polyacrylonitrile fiber and p-toluenesulfonamide and used for the preconcentration and separation for traces of Ru(III), Rh(III), Au(III) and Pd(IV) ions from waste water and ore sample solution. The synthesis of this fiber was simple and rapid. The results indicate that 100ngmL^–1 of these ions can be quantitatively enriched by the chelating fiber at a flow rate of 6mLmin^–1 and a pH of 4 and desorbed quantitatively with 20mL of 6molL^–1 HCl and 5% CS(NH₂)₂ solution at 50°C (with recovery>95%). A 50 to 1000-fold excess of Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, Fe³⁺, Cu²⁺, Zn²⁺, and Al³⁺ ions caused little interference in the concentration and determination of the analyzed ions. When the fiber had been reused twenty times, the recoveries of the ions enriched by the fiber were still over 96%. The saturated adsorption capacities of the fiber were in the range of 22–96mgg^–1. The relative standard deviation (RSD) of the method was between 0.70% and 0.84%. Recoveries of a standard added to actual samples were in range of 95–101%. The results obtained for these ions in real solution samples were basically in agreement with the given values, the average errors being below 5.0%. FT-IR spectra show that the existence of –SO₂–Ar, HN=C–NH–, O=C–NH– and –NH–SO₂ functional groups are verified in the chelating fiber. The experiments show that the method is rapid, precise, simple and convenient.     

Selective Solid Phase Extraction and Pre-Concentration of Heavy Metals from Seawater by Physically and Chemically Immobilized 4-Amino-3-Hydroxy-2-(2-Chlorobenzene)-Azo-1-Naphthalene Sulfonic Acid Silica Gel

4-Amino-3-hydroxy-2-(2-chlorobenzene)-azo-1-naphthalene sulfonic acid (AHCANSA) was used as a chelating modifier to improve the reactivity of the silica gel surface in terms of selective binding and extraction of heavy metal ions. The surface coverage values were found to be 0.488 and 0.473mmolg^–1 for the newly modified physically adsorbed silica gel phase (I) and chemically immobilized-AHCANSA phase (II), respectively. The modified silica gel phases (I, II) were tested for stability in different acidic buffer solutions (pH 1–6) and found to be highly resistant to hydrolysis and leaching by buffer solutions above pH 2. The application of these two phases as solid extractors for a series of mono-, di-, and tri-valent metal ions from aqueous solutions was also performed with different controlling factors such as the pH value of metal ion solutions and equilibrium shaking time. The mmolg^–1 metal capacity values determined by silica gel phases (I, II) were found to confirm high affinity and selectivity characters for binding with heavy metal ions such as Cr³⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Pb²⁺ in a range of 0.250–0.483. The tested alkali and alkaline earth metals, Na⁺, K⁺, Mg²⁺ and Ca²⁺, were found to exhibit little interaction and binding ability with the modified silica gel phases. The selectivity characters incorporated into the modified silica gel phases were further utilized and applied in solid phase extraction and pre-concentration of trace concentration levels (1.0µgmL^–1 and 2.00–2.50ngmL^–1) from real seawater samples. The percentage recovery values determined for Cr³⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Pb²⁺ were found to be in the range of 95.2–98.1±2.0–5.0%, and the pre-concentration recovery values for the same tested heavy metal ions were found to be in the range of 92.5–97.1±3.0–6.0% for the two newly modified silica gel phases with a pre-concentration factor of 500.Received December 20, 2002; accepted May 14, 2003 published online September 1, 2003     

Structural aspects of crown complexes with alkali and alkaline earth cations. Benzo-15-crown-5 as a discriminating macrocycle

X-ray structural results have been reviewed for the related M^z+ L _z ^– -B15C5 complexes where M^z+=Li⁺ to Cs⁺ and Mg²⁺ to Ba²⁺, L^–=2,4,6-trinitrophenolate (Picrate or Pic) and 3,5-dinitrobenzoate (Dnb), and B15C5=benzo-15-crown-5. These results combined with those for come MX_z-B15C5 (X=NCS^–, I^–, NO ₃ ^– , ClO ₄ ^– , BPh ₄ ^– ) complexes have revealed that B15C5 is a useful macrocycle with regard to the within-the-group and between-the-groups discriminations of M^z+ in the solid state.