Photosynthetic energy supply for NO3− assimilation in Scenedesmus

NO₃^?-dependent O₂ in synchronous Scenedesmus obtusiusculus Chod. in the absence of CO₂ is stoichiometric with NH₄⁺ excretion, indicating a close coupling of NO₃^? reduction to non-cyclic electron flow. Also in the presence of CO₂, NO₃^? stimulates O₂ evolution as manifested by an increase in the O₂/CO₂ ratio from 0.96 to 1.11. This quotient was increased to 1.36 by addition of NO₂^?, without competitive interaction with CO₂ fixation, indicating that the capacity for non-cyclic electron transport at saturating light is non-limiting for simultaneous reduction of NO₃^? and CO₂ at high rates. During incubation with NO₃^?+ CO₂, no NH₄⁺ is released to the outer medium, whereas during incubation with NO₂^?+ CO₂, excess NH₄⁺ is formed and excreted. NO₃^? uptake is stimulated by CO₂, and this stimulation is also significant when the cellular energy metabolism is restricted by moderate concentrations of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, whereas NO₃^? uptake in the absence of CO₂ is severely inhibited by the uncoupler. Also under energy-restricted conditions NO₃^? uptake is not competitive with CO₂ fixation. Antimycin A is inhibitory for NO₃^? uptake in the absence of CO₂, and there is no enhancement of NO₃^? uptake by CO₂ in the presence of antimycin A. It is assumed that the energy demand for NO₃^? uptake is met by energy fixed as triosephosphates in the Calvin cycle. Antimycin A possibly affects the transfer of reduced triose phosphates from the chloroplast to the cytoplasm. Active carbon metabolism also seems to exert a control effect on NO₃^? assimilation, inducing complete incorporation of all NO₃^? taken up into amino acids. This control effect is not functional when NO₂^? is the nitrogen source. Active carbon metabolism thus seems to be essential both for provision of energy for NO₃^? uptake and for regulation of the process.     

Uptake and loss of nitrite from the blood of rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L. in fresh water and in dilute sea water

The acute toxicity of nitrite (NO^?₂) to salmonids is strongly ameliorated by chloride (Cl^?) ions rendering it almost harmless in most fresh waters apart from those with low Cl^? content. In Cl^? poor fresh water external NO^?₂ is concentrated in the blood plasma until it is at approximately the same molar concentration as haemoglobin (about 8 mmol) and at this point most of the haemoglobin has been oxidized to methaemoglobin this being a contributory cause of death. Two theories are advanced to account for NO^?₂ concentration in the blood. The first supposes that gills are impermeable to NO^?₂ but allow its conjugate acid nitrous acid (HNO₂) to diffuse into the blood where it dissociates according to the blood pH value. Thus NO^?₂ will accumulate in the blood plasma if it has a higher pH value than the water. The second supposes that the Cl^? uptake mechanism in the freshwater gill has an affinity for NO^?₂ and accounts for the fact that NO^?₂ entry to the blood is suppressed when external Cl^? is present in significant amounts. The results also suggest that NO^?₂ and Cl^? behave similarly as diffusing ions. Thus NO^?₂ diffusion into the blood of seawater fish and from the blood of NO^?₂ loaded freshwater fish occurs at approximately the same rate as the corresponding Cl^? fluxes. Nitrite loss from seawater fish is thought to be mainly by diffusion although there is some evidence for the active Cl^? extrusion mechanism having a weak affinity for nitrite.     

Effect of pH on H-2H exchange,H2 production and H2 uptake,catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans

(1) The kinetics of isotope exchange catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans have been studied by measuring H²H, H₂ or ²H₂ produced when the enzyme catalyses the exchange between ²H₂ and H₂O or H₂ and ²H₂O. (2) In the ²H₂-H₂O system the measured rate of H₂ production was always higher than that of H²H. The $\text{H}{}_2\text{H}{}^2\text{H}$ ratio remained constant (about 1.70) in the protein concentration range 0.08–1.32 mg. The very rapid formation of H₂ with respect to H²H is consistent with the hypothesis of a heterolytic cleavage of ²H₂ into a deuteron and an enzyme hydride that can exchange with the solvent. (3) In the H₂-²H₂O system, the exchange rate was much lower than in the ²H₂-H₂O system, indicating a marked isotopic effect of ²H₂O. (4) The H-²H exchange activity, determined from the initial velocity of H²H formation, is optimal at pH 4.5. A second maximum of activity is observed at pH 8.3. The pH value of 4.5 is also the pH optimum for H₂ production while at pH 8.3–8.5 there is a maximum of H₂ oxidation activity. (5) In ordinary H₂O the K_m for hydrogen uptake estimated either from H₂ consumption or from benzyl viologen reduction was 0.06–0.07 μM for both H₂ and ²H₂ indicating a strong affinity of the enzyme for hydrogen at pH 8.3–8.5. Shifting from H₂O to ²H₂O does not affect the K_m of the enzyme for H₂ but lowers the V_max value about 10-fold. The K_m for benzyl viologen and methyl viologen was 0.08 and 2 mM, respectively.     

Staphylococcus aureus PerR Is a Hypersensitive Hydrogen Peroxide Sensor using Iron-mediated Histidine Oxidation

In many Gram-positive bacteria PerR is a major peroxide sensor whose repressor activity is dependent on a bound metal cofactor. The prototype for PerR sensors, the Bacillus subtilis PerR_BS protein, represses target genes when bound to either Mn²⁺ or Fe²⁺ as corepressor, but only the Fe²⁺-bound form responds to H₂O₂. The orthologous protein in the human pathogen Staphylococcus aureus, PerR_SA, plays important roles in H₂O₂ resistance and virulence. However, PerR_SA is reported to only respond to Mn²⁺ as corepressor, which suggests that it might rely on a distinct, iron-independent mechanism for H₂O₂ sensing. Here we demonstrate that PerR_SA uses either Fe²⁺ or Mn²⁺ as corepressor, and that, like PerR_BS, the Fe²⁺-bound form of PerR_SA senses physiological levels of H₂O₂ by iron-mediated histidine oxidation. Moreover, we show that PerR_SA is poised to sense very low levels of endogenous H₂O₂, which normally cannot be sensed by B. subtilis PerR_BS. This hypersensitivity of PerR_SA accounts for the apparent lack of Fe²⁺-dependent repressor activity and consequent Mn²⁺-specific repressor activity under aerobic conditions. We also provide evidence that the activity of PerR_SA is directly correlated with virulence, whereas it is inversely correlated with H₂O₂ resistance, suggesting that PerR_SA may be an attractive target for the control of S. aureus pathogenesis.     

A novel highly specific colorimetric fluorescent probe for the detection of nitrite in aqueous solution

Developing an effective method for the detection of nitrite (NO₂⁻) ions in the natural environment especially in environmental waters and soils is very necessary, since they will cause serious damage to human health once excess NO₂⁻ ions enters the human body. Therefore, a new colorimetric fluorescent probe NB-NO₂⁻ for determining NO₂⁻ ions was designed, which possesses good water-solubility and satisfactory selectivity over other common ions for NO₂⁻ ions. The addition of NO₂⁻ ions changed the color of solution from blue to colorless seen by the naked-eye. Furthermore, through test and calculation, the detection limit of the probe NB-NO₂⁻ is 129 nM. Based on the earlier excellent characteristics, the probe NB-NO₂⁻ was successfully used for monitoring NO₂⁻ ions in environmental waters and soils.     

Binding of divalent cations with poly (S-carboxymethyl-l-cysteine) and their effects on the polypeptide conformation

The effects of divalent ions on fully charged $\text{poly}\text{(S-}\text{carboxymethyl-}\text{l}\text{-cysteine}$) have been examined using circular dichroism for eight species: CuCl₂, CdCl₂, ZnCl₂, NiCl₂, CoCl₂, BaCl₂, CaCl₂, and MgCl₂. Five of them are effective inducers of β-form in the order: Cu²⁺Cd²⁺Zn²⁺Ni²⁺Co²⁺, in media with no salt added. However, the other three ions (Ba²⁺, Ca²⁺ and Mg²⁺), are not effective. Precipitation occurs when metal chlorides reach the vicinity of the equivalent point except for Ca²⁺ and Mg²⁺. Precipitation of the random coil form is slow, while that of the β-form is rapid. Addition of NcCl reduces the solubility of the β-form considerably. The pH value varies linearly with the logarithm of metal chloride concentration C_M for Ba²⁺, Ca²⁺ and Mg²⁺ ions, while nonlinear dependence of pH on log C_M is found for Cu²⁺, Ni²⁺ and Co²⁺ ions.     

Study of Pt(II)-cyclic amines complexes of the types cis- and trans-Pt(amine)2I2 and cis- and trans-Pt(amine)2(NO3)2 and their aqueous products by

Complexes of the types cis- and trans-Pt(amine)₂I₂ containing cyclic amines were synthesized and studied mainly by IR and multinuclear NMR spectroscopies. The compounds were converted to cis- and trans-Pt(amine)₂(NO₃)₂, which were also investigated. The hydrolysis and the aquation reactions of the latter compounds were then studied in D₂O in different conditions of pH. In acidic medium, the aqueous product is [Pt(amine)₂(D₂O)₂]²⁺ and for a few amines, [Pt(amine)₂(D₂O)(NO₃)]⁺ was detected. In basic pH, the main product is Pt(amine)₂(OD)₂ and Pt(amine)₂(OD)(NO₃) was detected for several compounds. In neutral pH, the cis isomers form between two and four species in fresh solutions. The most shielded species in ¹⁹⁵Pt NMR is the monoaqua-monohydroxo complex cis-[Pt(amine)₂(D₂O)(OD)]⁺ and the less shielded compound is the dihydroxo-bridged dimer [Pt(amine)₂(μ-OD)₂Pt(amine)₂]²⁺, which were observed for all the compounds. For a few amines, the monohydroxo-bridged dimer [Pt(D₂O)(amine)₂(μ-OD)Pt(OD)(amine)₂]²⁺ was detected and for cyclohexylamine, a fourth signal was assigned to a cyclic hydroxo-bridged trimer [(Pt(amine)₂(μ-OD))₃]³⁺. ¹⁹⁵Pt NMR spectroscopy has shown that the concentration of the monomer decreases with time, while the concentration of the dimers increases. Only one product was observed for the trans isomers in neutral pH. The signal was assigned to the monoaqua-monohydroxo species trans-[Pt(amine)₂(D₂O)(OD)]⁺. The ¹³C and ¹H NMR spectra of most of the complexes were measured. All the coupling constants ^2,3J(¹⁹⁵Pt-¹H) and ^2,3J(¹⁹⁵Pt-¹³C) are larger in the cis compounds than in the trans isomers.     

Vanadate inhibition of the Ca-ATPase from human red cell membranes

1. (1) VO₃⁻ combines with high affinity to the Ca²⁺-ATPase and fully inhibits Ca²⁺-ATPase and Ca²⁺-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca²⁺-dependent phosphoenzyme.

2. (2) VO₃⁻ blocks hydrolysis of ATP at the catalytic site. The sites for VO₃⁻ also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca²⁺-ATPase.

3. (3) The sites for VO₃⁻ show positive interactions in affinity with sites for Mg²⁺ and K⁺. This accounts for the dependence on Mg²⁺ and K⁺ of the inhibition by VO₃⁻. Although, with less effectiveness, Na⁺ substitutes for K⁺ whereas Li⁺ does not. The apparent affinities for Mg²⁺ and K⁺ for inhibition by VO₃⁻ seem to be less than those for activation of the Ca²⁺-ATPase.

4. (4) Inhibition by VO₃⁻ is independent of Ca²⁺ at concentrations up to 50 μM. Higher concentrations of Ca²⁺ lead to a progressive release of the inhibitory effect of VO₃⁻.

Insights into the Storage Mechanism of Layered VS2 Cathode in Alkali Metal‐Ion Batteries

VS₂ is one of the attractive layered cathodes for alkali metal‐ion batteries. However, the understanding of the detailed reaction processes and energy storage mechanism is still inadequate. Herein, the Li⁺/Na⁺/K⁺ insertion/extraction mechanisms of VS₂ cathode are elucidated on the basis of experimental analyses and theoretical simulations. It is found that the insertion/extraction behavior of Li⁺ is partially irreversible, while the insertion/extraction behavior of Na⁺/K⁺ is completely reversible. The detailed intermediates and final products (Li_0.33VS₂, LiVS₂, Na_0.5VS₂, NaVS₂, K_0.6VS₂, K_zVS₂, z > 0.6) during the discharging/charging processes are identified, indicating that VS₂ undergoes different phase transitions and solid–solution reactions in different battery systems, which have a great influence on the battery performance. Moreover, the diffusion of Na⁺ in VS₂ cathode is demonstrated to be much slower than that of Li⁺ and K⁺. Such mechanistic research provides a reference for in‐depth understanding of energy storage in layered transition metal sulfides/selenides.     

The J-coupling Restrained Molecular Mechanics (JrMM) Protocol - An Efficient Alternative for Deriving DNA Endocyclic Torsion Angle Constraints Part I: Correlation of Endocyclic Torsion Angles and Vicinal Torsion Angle φ1′2′

Abstract

An efficient alternative which makes use of the reliable ³J_1′2′. value to derive the endocyclic torsion angle constraints is proposed in this study. Based on the information embedded in the two plots, (i) the vicinal proton-proton J-couplings, ³J_1′2′., ³J_1′2″., ³J_2′3′., ³J_2”3′ and ³J_3′4′ against the pseudorotation phase angle, and (ii) ³J_1′2″, ³J_2′3′., ³J_2″3′ and ³J_3′4′ against ³J_1′2′; using the calculated J-couplings obtained for a range of sugar geometries of deoxyribose ring in nucleosides and nucleotides encountered along the pseudorotation itinerary [J. van Wijk, B.D. Huckriede, J.H. Ippel and C. Altona, Methods Enzymol. 211, 286–306 (1992)], it is suggested that the vicinal ³J_1′2′ possesses structural information other than the vicinal torsion angle φ_1′2′. This study is divided into two parts. In Part I, a correlation diagram between the endocyclic torsion angles v_i (i=0,1,2,3,4) and the restrained vicinal torsion angle φ_1′2′ is obtained through the use of the J-coupling restrained molecular mechanics (JrMM) protocol. The established φ_1′2′.-v_i correlation shows v_i can be deduced from the reliable ³J_1′2′. value and it forms the basis for developing an alternative protocol to derive endocyclic torsion angle constraints. In Part II of this series, extensive testing demonstrating the validity of the JrMM protocol to derive V_i for defining the sugar geometry of solution DNA molecules is presented.     

Hydropropidine: A novel,cell-impermeant fluorogenic probe for detecting extracellular superoxide

Here we report the synthesis and characterization of a membrane-impermeant fluorogenic probe, hydropropidine (HPr⁺), the reduction product of propidium iodide, for detecting extracellular superoxide (O₂^•−). HPr⁺ is a positively charged water-soluble analog of hydroethidine (HE), a fluorogenic probe commonly used for monitoring intracellular O₂^•−. We hypothesized that the presence of a highly localized positive charge on the nitrogen atom would impede cellular uptake of HPr⁺ and allow for exclusive detection of extracellular O₂^•−. Our results indicate that O₂^•− reacts with HPr⁺ (k=1.2×10⁴ M⁻¹ s⁻¹) to form exclusively 2-hydroxypropidium (2-OH-Pr²⁺) in cell-free and cell-based systems. This reaction is analogous to the reaction between HE and O₂^•− (Zhao et al., Free Radic. Biol. Med. 34:1359–1368; 2003). During the course of this investigation, we also reassessed the rate constants for the reactions of O₂^•− with HE and its mitochondria targeted analog (Mito-HE or MitoSOX Red) and addressed the discrepancies between the present values and those reported previously by us. Our results indicate that the rate constant between O₂^•− and HPr⁺ is slightly higher than that of HE and O₂^•− and is closer to that of Mito-HE and O₂^•−. Similar to HE, HPr⁺ undergoes oxidation in the presence of various oxidants (peroxynitrite-derived radicals, Fenton's reagent, and ferricytochrome c) forming the corresponding propidium dication (Pr²⁺) and the dimeric products (e.g., Pr²⁺-Pr²⁺). In contrast to HE, there was very little intracellular uptake of HPr⁺. We conclude that HPr⁺ is a useful probe for detecting O₂^•− and other one-electron oxidizing species in an extracellular milieu.     

PHOTOSYNTHESIS AND CALCIFICATION BY EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) AS A FUNCTION OF INORGANIC CARBON SPECIES

To test the possibility of inorganic carbon limitation of the marine unicellular alga Emiliania huxleyi (Lohmann) Hay and Mohler, its carbon acquisition was measured as a function of the different chemical species of inorganic carbon present in the medium. Because these different species are interdependent and covary in any experiment in which the speciation is changed, a set of experiments was performed to produce a multidimensional carbon uptake scheme for photosynthesis and calcification. This scheme shows that CO₂ that is used for photosynthesis comes from two sources. The CO₂ in seawater supports a modest rate of photosynthesis. The HCO is the major substrate for photosynthesis by intracellular production of CO₂ (HCO+ H⁺→ CO₂+ H₂O → CH₂O + O₂). This use of HCO is possible because of the simultaneous calcification using a second HCO, which provides the required proton (HCO+ Ca²⁺→ CaCO₃+ H⁺). The HCO is the only substrate for calcification. By distinguishing the two sources of CO₂ used in photosynthesis, it was shown that E. huxleyi has a K_½ for external CO₂ of “only” 1.9 ± 0.5 μM (and a V_max of 2.4 ± 0.1 pmol·cell⁻¹·d⁻¹). Thus, in seawater that is in equilibrium with the atmosphere ([CO₂]= 14 μM, [HCO]= 1920 μM, at fCO₂= 360 μatm, pH = 8, T = 15° C), photosynthesis is 90% saturated with external CO₂. Under the same conditions, the rate of photosynthesis is doubled by the calcification route of CO₂ supply (from 2.1 to 4.5 pmol·cell⁻¹·d⁻¹). However, photosynthesis is not fully saturated, as calcification has a K_½ for HCO of 3256 ± 1402 μM and a V_max of 6.4 ± 1.8 pmol·cell⁻¹·d⁻¹. The H⁺ that is produced during calcification is used with an efficiency of 0.97 ± 0.08, leading to the conclusion that it is used intracellularly. A maximum efficiency of 0.88 can be expected, as NO uptake generates a H⁺ sink (OH⁻ source) for the cell. The success of E. huxleyi as a coccolithophorid may be related to the efficient coupling between H⁺ generation in calcification and CO₂ fixation in photosynthesis.     

CO2-concentrating mechanisms: a direct role for thylakoid lumen acidification?

A testable mechanism of CO₂ accumulation in photolithotrophs, originally suggested by Pronina & Semenenko, is quantitatively analysed. The mechanism involves (as does the most widely accepted hypothesis) the delivery of HCO₃^? to the compartment containing Rubisco. It differs in proposing subsequent HCO₃^? entry (by passive uniport) to the thylakoid lumen, followed by carbonic anhydrase activity in the lumen; uncatalysed conversion of HCO₃^? to CO₂, even at the low pH of the lumen, is at least 300 times too slow to account for the rate of inorganic C acquisition. Carbonic anhydrase converts the HCO₃^? to CO₂ at the lower pH maintained in the illuminated thylakoid lumen by the light-driven H⁺ pump, generating CO₂ at 10 times or more the thylakoid HCO₃^? concentration. Efflux of this CO₂ can suppress Rubisco oxygenase activity and stimulate carboxylase activity in the stroma. This mechanism differs from the widely accepted hypotheses in the required location of carbonic anhydrase, i.e. in the thylakoid lumen rather than the stroma or pyrenoid, and in the need for HCO₃^? influx to thylakoids. The capacity for anion (assayed as Cl^?) entry by passive uniport reported for thylakoid membranes is adequate for the proposed mechanism; if the Cl^? channel does not transport HCO₃^?, HCO₃^? entry could be by combination of the Cl^? channel with a Cl^? HCO₃^? antiporter. This mechanism is particularly appropriate for organisms which lack overt accumulation of total inorganic C in cells, but which nevertheless have the gas exchange characteristics of an organism with a CO₂-concentrating mechanism.     

A trigonal-prismatic Ag(I) complex and an octahedral Cu(II) complex incorporating with oxamide Ni(II) complex ligand: Syntheses, crystal structures and oxidative nuclease activities

A mononuclear macrocyclic complex Ni^IIL^3a (L^3a = dianion of 2,3-dioxo-5,6:13,14-dibenzo-9,10-cyclohexyl-7,12-bis(methoxycarbonyl)-1,4,8,11-tetraazacyclotetradeca-7,11-diene), which shows high DNA cleavage activity in the presence of H₂O₂, was reported in our previous work. Considering that many systems for natural enzyme-mediated DNA cleavage contain two or more metal active sites, two new trinuclear complexes [Cu(NiL^3a)₂(dca)₂]·2CH₃OH (abbreviated as Cu(NiL^3a)₂) and [Ag(NiL^3a)₂(NO₃)]·2CH₃OH·0.5H₂O (abbreviated as Ag(NiL^3a)₂) were synthesized in this work, where dca is the dicyanamide. The complexes were structurally characterized by single crystal X-ray analysis. The central Cu(II) or Ag(I) atom is linked to two [NiL^3a] ligands by oxamido bridges forming a trinuclear structure. In Cu(NiL^3a)₂, the central Cu(II) ion is in an octahedral coordination geometry. Whereas in Ag(NiL^3a)₂, the central Ag(I) ion is in a rarely reported trigonal-prismatic coordination geometry. The DNA cleavage behavior of the complexes in the presence of H₂O₂ was studied in detail. Comparing with the NiL^3a, the trinuclear complex Ag(NiL^3a)₂ nearly has no ability to cleave DNA, whereas Cu(NiL^3a)₂ is a much better DNA cleavage agent. Cu(NiL^3a)₂ can efficiently convert supercoiled DNA to nicked DNA with a rate constant of 0.074 ± 0.002 min⁻¹ when 40 μM Cu(NiL^3a)₂ and 0.6 mM H₂O₂ are used. The cleavage mechanism between the complex Cu(NiL^3a)₂ and plasmid DNA is likely to involve singlet oxygen as reactive oxygen species. Circular dichroism (CD) and fluorescence spectroscopy indicate that both Cu(NiL^3a)₂ and NiL^3a bind to DNA by a groove binding mode, and the binding between Cu(NiL^3a)₂ and DNA is much stronger than that between NiL^3a and DNA. The present results may provide some information for the design of efficient multinuclear artificial nucleases.     

Determination of H2O2-dependent generation of singlet oxygen from human saliva with a novel chemiluminescence probe

Singlet oxygen (¹O₂) has been shown to play an important role in salivary defense system, but its generation process and level from human saliva remain uncertain due to the lack of a reliable detection method. We have previously reported 4,4′(5′)-bis[2-(9-anthryloxy)ethylthio]tetrathiafulvalene (BAET) as a novel chemiluminescence probe for ¹O₂. In this work, the probe is successfully used to characterize H₂O₂-dependent generation of ¹O₂ from saliva in real time. However, the yield of ¹O₂ is found to be very low, for example, being about 0.13 nmol from 200 μL saliva in the presence of 1 mM of hydrogen peroxide over a 5-s reaction period. The result is also compared with that obtained with another ¹O₂ probe 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), demonstrating that, besides ¹O₂, the other reactive oxygen species such as hydroxyl radical may also be involved in the reaction of saliva with H₂O₂. Furthermore, the present study shows that the selectivity of BAET for ¹O₂ is much higher than that of CLA and thus BAET is highly suited for the detection of ¹O₂ in the presence of other reactive oxygen species in biological systems.     

Density Functional Geometry Optimization and Energy Calculations of Calcium(II)-Triphosphate Complexes. Polyphosphates as Possible Dissolving Agents for Calcium Pyrophosphate Dihydrate Crystals in Chondrocalcinosis Disease

Abstract

Geometry optimizations and energy calculations have been carried out via molecular orbital methods at the density functional B3LYP/LANL2DZ level on the molecules PO₃ ^?, OPO₃ ^3-, HOPO₃ ^2-, CH₃OPO₃ ^2-, H(CH₃OPO₃ ^?, O(PO₃)₂ ^4-, HO(PO₃)₂ ^3-, CH₂ (PO₃)₂ ^4-, (CH₃OPO₂)O(PO₃)^3-, O(PO₃)₃ ^5-, HO(PO₃)₃ ^4-, (PO₃)₃ ^3-, (CH₃OPO₂)O(PO₃)₂ ^3-, [Mg{O(PO₃)₂}]^2-, [Ca{O(PO₃)₂}]^2-, [Ca{CH₂ (PO₃)₂}]^2-, [Ca{CH₃OPO₂)O(PO₃)}]^?, [Ca(PO₃)₃]^?, [Ca{O(PO₃)₃}]^3-, and [Ca{CH₃OPO₂)O(PO₃)₂}]^2- with the aim to find reliable and easily accessible computational methods to simulate some phosphate-containing molecules of importance for the living cells and to study the energetics for protonation and metal-complex formation reactions. The analysis is part of a general investigation on phosphate-containing molecules as potential dissolving agents for calcium pyrophosphate dihydrate (CPPD) crystals which deposit in certain articular diseases. The basis set was expanded to 6–31G** for the P atoms for all the molecules investigated and to 6–31G* for the O atoms for OPO₃ ^3-. Calculations at the semi- empirical MNDO/d level were also carried out for comparison purposes on the free ligand molecules and on [Mg{O(PO₃)₂}]^2-. The density functional analysis reproduced well the geometry found at the solid state via X-ray diffraction. The analyses of the geometrical parameters and the total electronic energy of the molecules shows that O(PO₃)₂ ^4- and other di- and tri-phosphates are versatile ligands for divalent metal ions like Ca²⁺. The computed P-O-P bond angle for free O(PO₃)₃ ^4- is 180° and the conformation of the two PO₃ ^? groupings is staggered along the P…P vector. The linear arrangement for P-O-P is assisted by P-Oπ interactions. The bending of the P-O-P angle when accompanied by a slight P-O(b) elongation requires a very small amount of energy; 4.65 kcal/mol to pass from 180 to 140°, as calculated at the DFT level. The computed Ca-O and Mg-O bond distances for [M{O(PO₃)₂}]^2- are 2.378 and 2.079Å, when the metal ions link two oxygen atoms from each PO₃ group. The computed Ca-O bond lengths for [Ca{CH₃OPO₂)O(PO₃)}]^? are 2.482 (P_αO₂) and 2.358Å (P_βO₂), showing a significant lengthening for Ca-OP_α, when compared to the pyrophosphate derivative. The Ca-O bond lengths for [Ca{O(PO₃)₃}]^3- and [Ca{CH₃OPO₂)O(PO₃)2}]^2- are 2.251Å and 2.525 (P_αO₂), 2.407 and 2.338 (P_βO₂), and 2.251 and 2.228Å (P_γO₂), showing a shortening for the Ca-OPγ bond upon methylation. The (P_β)O-P_γ bond length increases significantly (0.09 Å) upon Ca(II) coordination to (CH₃OPO₂)O(PO₃)₂ ^4- via all the three PO₃ groups. This latter result suggests that metal complexes of linear organic-triphosphates have a larger tendency to release the P_γO₃ group when compared to the free ligand molecules. The electronic contribution to the energy of the complex formation reaction for [Ca{CH₂ (PO₃)₂}]^2- is only slightly higher (some 1.8 kcal) than that for [Ca{O(PO₃)₂}]^2-; but is much higher (some 63 kcal) than that relevant to the formation of [Ca{CH₃OPO₂)O(PO₃)₂}]^2-. Therefore the linear methyltriphosphates (and reasonably nucleoside triphosphates) are predicted to form metal complexes which are less stable than diphosphonate or inorganic diphosphate ligands, at least in the case the organic moiety does not have any appreciable coordination ability toward the metal. The energy of complex formation for [Ca{O(PO₃)₃}]^3- is much higher than that for [Ca{O(PO₃)₂}]^2- and [Ca(PO₃)₃}]^? (some 116 and 345 kcal, respectively). This is in part a rationale for the high dissolving ability (DA) toward CPPD crystals, shown by solutions of pentasodium tripolyphosphate. DA values of 65.4±3.0 μg Ca/mL were detected, via atomic absorption spectroscopy, in solutions of Na₅{O(PO₃)₃} 0.06 M after digestion (1.00 h, 37°C, pH 7.4) in the presence of CPPD crystals. Efficient non-toxic dissolving agents for CPPD crystals are needed to cure chondrocal-cinosis desease. The semi-empirical computations gave correct overall optimized structures and conformations for, at least, the free not-substituted ligands and the relevant Mg(II)-com- plexes. The optimized structure for [Mg{O(PO₃)₂}]^2- has computed P-O(t) and P-O(d) bond distances of 1.503 and 1.598Å, in perfect agreement with the results from DFT calculations.     

Stability of metal ion complexes formed with methyl phosphate and hydrogen phosphate

 The acidity constants of methyl phosphoric acid, CH₃OPO(OH)₂, and orthophosphoric acid, HOPO(OH)₂, and the stability constants of the 1 : 1 complexes formed between Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, or Cd²⁺ and methyl phosphate, CH₃OPO₃ ^2–, or hydrogen phosphate, HOPO₃ ^2–, were determined by potentiometric pH titration in aqueous solution (25  °C;I = 0.1 M, NaNO₃). On the basis of previously established log K versus pK _a straight-line plots for the complexes of simple phosphate monoesters and phosphonate derivatives, R-PO₃ ^2–, where R is a noncoordinating residue, it is shown that the stability of the M(CH₃OPO₃) complexes is solely determined (as one might expect) by the basicity of the –PO₃ ^2– residue. It is emphasized that the mentioned reference lines may also be used to reveal increased complex stabilities, for example, for certain complexes formed with 8-quinolyl phosphate the occurrence of 7-membered chelates can be proven in this way; the same procedure is also applicable to complexes of nucleotides, etc. The M(HOPO₃) complexes are slightly more stable (on average by 0.08 log unit) than it is expected from the basicity of HPO₄ ^2–; this observation is attributed to a more effective solvation, including hydrogen bonding, than is possible with CH₃OPO₃ ^2– species. Received: 9 November 1995 / Accepted: 5 February 1996     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.     

Leukotriene D4-induced Ca2+ Mobilization in Ehrlich Ascites Tumor Cells

Stimulation of Ehrlich ascites tumor cells with leukotriene D₄ (LTD₄) within the concentration range 1–100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca²⁺ concentration, [Ca²⁺] _i . The Ca²⁺ peak time, i.e., the time between addition of LTD₄ and the highest measured [Ca²⁺] _i value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration of LTD₄ (100 nm), the highest measured increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium is 260 ± 14 nm and the EC₅₀ value for LTD₄-induced Ca²⁺ mobilization is estimated at 10 nm. Neither the peptido-leukotrienes LTC₄ and LTE₄ nor LTB₄ are able to mimic or block the LTD₄-induced Ca²⁺ mobilization, hence the receptor is specific for LTD₄. Removal of Ca²⁺ from the experimental buffer significantly reduces the size of the LTD₄-induced increase in [Ca²⁺] _i . Furthermore, depletion of the intracellular Ins(1,4,5)P₃-sensitive Ca²⁺ stores by addition of the ER-Ca²⁺-ATPase inhibitor thapsigargin also reduces the size of the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-containing medium, and completely abolishes the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells suspended in Ca²⁺-free medium containing EGTA. Thus, the LTD₄-induced increase in [Ca²⁺] _i in Ehrlich cells involves an influx of Ca²⁺ from the extracellular compartment as well as a release of Ca²⁺ from intracellular Ins(1,4,5)P₃-sensitive stores. The Ca²⁺ peak times for the LTD₄-induced Ca²⁺ influx and for the LTD₄-induced Ca²⁺ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35 min in six out of eight experiments, respectively. Stimulation with LTD₄ also induces a transient increase in Ins(1,4,5)P₃ generation in the Ehrlich cells, and the Ins(1,4,5)P₃ peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4,5)P₃ content seems to increase before the LTD₄-induced Ca²⁺ release from the intracellular stores but after the LTD₄-induced Ca²⁺ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD₄-induced increase in Ins(1,4,5)P₃ as well as the LTD₄-induced increase in [Ca²⁺] _i , indicating that a U73122-sensitive phospholipase C is involved in the LTD₄-induced Ca²⁺ mobilization in Ehrlich cells. The LTD₄-induced Ca²⁺ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD₄-activated Ca²⁺ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion, LTD₄ acts in the Ehrlich cells via a specific receptor for LTD₄, which upon stimulation initiates an influx of Ca²⁺, through yet unidentified Ca²⁺ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P₃ formation and finally release of Ca²⁺ from the intracellular Ins(1,4,5)P₃-sensitive stores. Received: 9 February 1996/Revised: 15 August 1996