Strategies for the measurement of the inhibitory effects of thymidine analogs on the activity of thymidylate synthase in intact murine leukemia L1210 cells

Two strategies have been pursued to monitor the inhibition of thymidylate (dTMP) synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) by thymidine (dThd) analogs in intact murine leukemia L1210 cells. The first method was based on the determination of tritium release from 2'-deoxy[5-3H]uridine [( 5-3H]dUrd) or 2'-deoxy[5-3H]cytidine [( 5-3H]dCyd); the second method was based on an estimation of the amount of dCyd incorporated into DNA as dTMP. The validity of these procedures was assessed by evaluating the inhibition of thymidylate synthase in murine leukemia L1210 cells by a series of 18 dThd analogs. There was a strong correlation between the inhibitory effects of the dThd analogs on the proliferation of L1210 cells on the one hand, and (i) their inhibitory effects on tritium release from [5-3H]dCyd (r = 0.926) and (ii) their inhibitory effects on the incorporation of dCyd into DNA dTMP (r = 0.921), on the other hand. Evaluation of tritium release from [5-3H]dCyd proved to be the most convenient method that has been described so far to measure thymidylate synthase activity and to follow the inhibitory effects of thymidylate synthase inhibitors in intact L1210 cells, since this method is rapid and very sensitive, and since it proved superior to the evaluation of tritium release from [5-3H]dUrd because it circumvents possible interactions of the inhibitors with thymidine kinase activity.     

Thymidylate synthetase-deficient mouse FM3A mammary carcinoma cell line as a tool for studying the thymidine salvage pathway and the incorporation of thymidine analogues into host cell DNA.

A thymidylate (dTMP) synthetase-deficient murine mammary carcinoma cell line (FM3A/TS-), auxotrophic for thymidine (dThd), proved extremely useful for studying the dependence of cell growth on the exogenous supply of dThd, the relation between cell growth and DNA synthesis, and the ability of a series of 25 5-substituted 2'-deoxyuridines (dUrd) to substitute for dThd in sustaining cell growth. FM3A/TS-cells did not proliferate unless dThd was supplied to the cell culture medium. The 5-halogenated dUrd derivatives 5-chloro-dUrd, 5-bromo-dUrd and 5-iodo-d Urd also sustained FM3A/TS- cell growth. The extents of incorporation of [methyl-3H]dThd and 5-iodo-[6-3H]dUrd into DNA were closely correlated with their stimulatory effects on FM3A/TS- cell growth. This suggests that the stimulatory effects of the dUrd analogues on the growth rate of FM3A/TS- cells may be considered as evidence for their incorporation into host cell DNA. Based on this premise it is postulated that, in addition to 5-chloro-dUrd, 5-bromo-dUrd, 5-iodo-dUrd and dThd itself, the following dThd analogues are also incorporated into FM3A/TS- cell DNA (in order of the extent to which they are incorporated): 5-hydroxy-dUrd greater than 5-propynyloxy-dUrd greater than 5-ethyl-dUrd greater than 5-ethynyl-dUrd approximately 5-vinyl-dUrd. Thus, the dTMP synthetase-deficient FM3A/TS- cell line represents a unique system to dissociate the de novo and salvage pathways of dTMP biosynthesis and to distinguish those dUrd analogues that are incorporated into DNA from those that are not.     

Heavy metal-nucleotide interactions. IX. Raman difference spectroscopic studies on the binding of CH3Hg(II) to 1-methylthymine, thymidine-5'-monophosphate, DNA models and native DNA.

Raman spectra have been obtained for dTMP and its complex with CH3Hg (II) in aqueous solution as a function of pH. Difference spectroscopy is employed to increase the sensitivity of the Raman technique. The binding reaction is essentially quantitative from pH 3 to 9, and the value of the equilibrium constant for CH3HgOH2+ + dThd in equilibrium CH3Hg(dThdH--1) + H30+ is estimated from intensity measurements to be 0.6 in reasonable agreement with an earlier value based upon uv spectrophotometric data. Binding is to N(3) with substitution of CH3Hg+ for the proton. A similar reaction occurs with 1-MeThy. Raman spectra for aqueous and crystalline 1-MeThy and for the complex CH3Hg(1-MeThyH--1) are reported. The spectrum of crystalline Hg(1-MeThyH--1)2, for which the crystal structure is known, also was obtained for comparison. Raman difference spectroscopy was used to confirm that CH3Hg (II) binds to N(3) of dTMP and N(1) of GMP at r = 0.2 (MeHg+: phosphate) ratios with mixtures of GMP + CMP + AMP + dTMP. In contrast, native calf thymus DNA does not appear to bind CH3Hg(II) at these sites at r = 0.15, although no significant amount of free CH3HgOH is present. With r = 0.3, extensive binding occurs both to the Thy and Gua bases. Raman difference spectroscopy is a valuable technique for studying the binding of ions and molecules to polynucleotides in moderately dilute aqueous solution.     

Reversible permeabilization of lymphocytes destroys the incorporation of deoxythymidine but not of deoxycytidine

The total uptake, phosphorylation and incorporation of thymidine (dThd) and deoxycytidine (dCyd) were compared in intact and reversibly permeabilized human tonsillar lymphocytes. The total uptake of [3H]dThd was lower than that of [5-3H]dCyd, but almost all of [3H]dThd was incorporated into DNA. However, the main part of [5-3H]dCyd taken up by the lymphocytes was found in the pool as phosphorylated nucleoside (55%), and only a smaller part (13%) was incorporated into DNA. Phosphorylated nucleosides were determined by DEAE-cellulose sheets in the ethanol-soluble fraction of the cells. The reversible permeabilization of lymphocytes by Dextran T-150 destroys totally the [3H]dThd incorporation, while [5-3H]dCyd incorporation decreased only to 60% of intact cells. During permeabilization the phosphorylation of both nucleosides increased severalfold. After permeabilization all [3H]dThd was in dTMP form, while [5-3H]dCyd was also found in dCDP (3%) and dCTP (38%) form. In the meanwhile, 22% of thymidine kinase, 63% of deoxycytidine kinase and 98% of DNA polymerase activity were measured in permeabilized cells as compared to intact cells. The results suggest different relationships between the lymphocyte plasma membrane and the salvage pathways of the two pyrimidine nucleosides.     

Cell cycle regulation and novel structural features of thymidine kinase,an essential enzyme in Trypanosoma brucei

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ‐phosphate of ATP to 2′‐deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C‐terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine‐derived nucleotides. In addition, we report the X‐ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.     

Monovalent cation size and DNA conformational stability

The effect of monovalent cations on the thermal stability of a small model DNA hairpin has been measured by capillary electrophoresis, using an oligomer with 16 thymine residues as an unstructured control. The melting temperature of the model hairpin increases approximately linearly with the logarithm of increasing cation concentration in solutions containing Na(+), K(+), Li(+), NH(4)(+), Tris(+), tetramethylammonium (TMA(+)), or tetraethylammonium (TEA(+)) ions, is approximately independent of cation concentration in solutions containing tetrapropylammonium (TPA(+)) ions, and decreases with the logarithm of increasing cation concentration in solutions containing tetrabutylammonium (TBA(+)) ions. At constant cation concentration, the melting temperature of the DNA model hairpin decreases in the order Li(+) ～ Na(+) ～ K(+) > NH(4)(+) > TMA(+) > Tris(+) > TEA(+) > TPA(+) > TBA(+). Isothermal studies indicate that the decrease in the hairpin melting temperature with increasing cation hydrophobicity is not due to saturable, site-specific binding of the cation to the random coil conformation, but to the concomitant increase in cation size with increasing hydrophobicity. Larger cations are less effective at shielding the charged phosphate residues in B-form DNA because they cannot approach the DNA backbone as closely as smaller cations. By contrast, larger cations are relatively more effective at shielding the phosphate charges in the random coil conformation, where the phosphate-phosphate distance more closely matches cation size. Hydrophobic interactions between alkylammonium ions interacting electrostatically with the phosphate residues in the coil may amplify the effect of cation size on DNA thermal stability.     

Biochemical evidence for the existence of thymidylate synthase in the obligate intracellular parasite Chlamydia trachomatis.

Since eucaryotic cell-derived thymidine or thymidine nucleotides are not incorporated into Chlamydia trachomatis DNA, we hypothesized that C. trachomatis must obtain dTTP for DNA synthesis by converting dUMP to dTMP. In most cells, this reaction is catalyzed by thymidylate synthase (TS) and requires 5,10-methylenetetrahydrofolate as a cofactor. We used C. trachomatis serovar L2 and a mutant CHO K1 cell line with a genetic deficiency in folate metabolism as a host for chlamydial growth. This cell line lacks a functional dihydrofolate reductase (DHFR) gene and, as a result, is unable to carry out de novo synthesis of dTTP. C. trachomatis inclusions form normally when DHFR- cells are starved for thymidine 24 h prior to and during the course of infection. When [6-3H]uridine is used as a precursor to label C. trachomatis-infected CHO DHFR- cells, radiolabel is readily incorporated into chlamydia-specific DNA. When DNA from [6-3H]uridine-labelled infected cultures is acid hydrolyzed and subjected to high-performance liquid chromatography analysis, radiolabel is detected in thymine and cytosine nucleobases. By using the DHFR- cell line as a host and [5-3H]uridine as a precursor, we could monitor intracellular C. trachomatis TS activity simply by following the formation of tritiated water. There is a good correlation between in situ TS activity and DNA synthesis activity during the chlamydial growth cycle. In addition, both C. trachomatis-specific DNA synthesis and 3H2O release are inhibited by exogenously added 5-fluorouridine but not by 5-fluorodeoxyuridine. Finally, we demonstrated in vitro TS activity in crude extracts prepared from highly purified C. trachomatis reticulate bodies. The activity is dependent on the presence of methylenetetrahydrofolic acid and can be inhibited with 5-fluoro-dUMP. Taken together, these results indicate that C. trachomatis contains a TS for the synthesis of dTMP.     

Sugar complexation with calcium ion. Crystal structure and FT-IR study of a hydrated calcium chloride complex of D-ribose

The single crystal structure of CaCl(2).C(5)H(10)O(5).3H(2)O was determined with M(r)=315.16, a=7.537(3), b=11.426(5), c=15.309(6) A, beta=90 degrees, V=1318.3(9) A(3), P2(1)2(1)2(1), Z=2, mu=0.71073 A and R=0.0398 for 2322 observed reflections. The ribose moiety of the complex exists as a furanose with alpha-D configuration. All five oxygen atoms of the ribose molecule are involved in calcium binding. Each calcium ion is shared by two such sugar molecules, coordinating through O(1), O(2), O(3) of one molecule and O(4) and O(5) of the other. The C-C, O-H, C-O and C-O-H vibrations are shifted and the relative intensities changed in the complex IR spectrum, corresponding to the changes in bond distances and angles of the sugar structure. All the hydroxyl groups, water molecules and chloride ions are involved in forming an extensive hydrogen-bond network of O-H...Cl...O-H structure, and the chloride ions play an important role in the crystal packing.     

alpha-DNA. I. Synthesis, characterization by high field 1H-NMR, and base-pairing properties of the unnatural hexadeoxyribonucleotide alpha-[d(CpCpTpTpCpC)] with its complement beta-[d(GpGpApApGpG)].

The novel deoxyribonucleotide alpha-[d(CpCpTpTpCpC)] and its complement beta-[d(GpGpApApGpG)] were synthesized by the phosphotriester method. 1H-NMR-NOE examination of the alpha-hexamer revealed that the cytosine and thymine bases appear to adopt anti conformations in this strand. In addition the deoxyribose of the thymidine moieties may adopt average conformations approximating to C3'-endo while the cytidine furanose groups are close to C2'-endo conformations. Both hyperchromicity in thermal melting and detection of base paired imino protons in 1H-NMR studies in H2O provide evidence for the annealing of alpha-d[CCTTCC] with its complement beta-d[GGAAGG] in potassium phosphate buffer pH 7.1 containing 10 mM magnesium chloride. Under these conditions thermal melting begins at 38 degrees C and its complete at approximately 45 degrees C. NOE experiments do not permit a decision on the polarity of annealing (predicted to be parallel) for this particular pair of sequences.     

New, partially hydrolyzable synthetic analogues of lecithin, phosphatidyl ethanolamine, and phosphatidic acid

The synthesis of two new synthetic analogues of lecithin, two of phosphatidyl ethanolamine ("cephalin"), and one new phosphatidic acid analogue is described. They comprise one of each of the following types: the "isosteric" diether lecithin and cephalin analogues ROCH(2)CH(OR)- CH(2)CH(2)P(O) (O(-))OCH(2)CH(2)N(+)R'(3) (R = C(18)H(37); R' = H or CH(3)); and the "hydrocarbon" analogues of phosphatidic acid, lecithin, and cephalin, C(17)H(35)CH(2)CH(C(18)H(37))CH(2)P(O)(R) = (R'); [R = R' = OH; R = O(-), R' = OCH(2)CH(2)N(+)(CH(3))(3); and R = O(-), R' = OCH(2)CH(2)N(+)H(3)]. Infrared spectra and other properties of these compounds are described.     

Nucleic acid metabolism in yeast, V. Excretion of thymidylate

It is shown that highly efficient utilisers of exogenous dTMP of the yeast Saccharomyces cerevisiae are able to excrete the nucleotide with similar efficiency. Strains Pi-repressible in acid phosphatase/nucleotidase excrete dTMP at extracellular high Pi; strains constitutive for this enzymic activity excrete dThd. Excretion of thymidylate and dThd, unlike uptake of exogenous dTMP, seems to be unaffected by the extracellular pH, by the extracellular presence of dTMP, and to be rather independent of the extracellular presence of a metabolisable carbohydrate such as D(+)-glucose. A model of the yeast dTMP-incorporation principle (TIP) is presented suggesting that it is also responsible for export of endogenous thymidylate.     

Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

Involvement of the H3O+-Lys-164 -Gln-161-Glu-345 charge transfer pathway in proton transport of gastric H+,K+-ATPase

Gastric H(+),K(+)-ATPase is shown to transport 2 mol of H(+)/mol of ATP hydrolysis in isolated hog gastric vesicles. We studied whether the H(+) transport mechanism is due to charge transfer and/or transfer of hydronium ion (H(3)O(+)). From transport of [(18)O]H(2)O, 1.8 mol of water molecule/mol of ATP hydrolysis was found to be transported. We performed a molecular dynamics simulation of the three-dimensional structure model of the H(+),K(+)-ATPase alpha-subunit at E(1) conformation. It predicts the presence of a charge transfer pathway from hydronium ion in cytosolic medium to Glu-345 in cation binding site 2 (H(3)O(+)-Lys-164 -Gln-161-Glu-345). No charge transport pathway was formed in mutant Q161L, E345L, and E345D. Alternative pathways (H(3)O(+)-Gln-161-Glu-345) in mutant K164L and (H(3)O(+)-Arg-105-Gln-161-Gln-345) in mutant E345Q were formed. The H(+),K(+)-ATPase activity in these mutants reflected the presence and absence of charge transfer pathways. We also found charge transfer from sites 2 to 1 via a water wire and a charge transfer pathway (H(3)O(+)-Asn-794 -Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H(2)O in sites 2 and 1, the H(2)O comes from cytosolic medium, and H(3)O(+) in the sites are transported into lumen during the conformational transition from E(1)PtoE(2)P.     

Adsorption equilibria at interface separating electrolyte solution and phosphatidylcholine-stearylamine liposome membrane

The effect of the pH of an electrolyte solution on the electric surface charge of the liposome membrane was studied. The membrane of vesicles contained egg phosphatidylcholine (PC) with different proportions of stearylamine (ST). The surface charge density of the membrane was determined as a function of pH from electrophoretic mobility measurements. A six equilibria model describing the solution ions adsorption on the PC-ST liposome membrane surface was presented in this paper. The knowledge of the association constants of the -PO(-) and -N(+)CH(3)(3) groups of PC with H(+), OH(-), Na(+), Cl(-) ions: K(A(1)H), K(B(1)OH), K(A(1)Na), K(B(1)Cl), that had been presented earlier, allowed to determine the association constants of the -N(+)H(3) group of ST with OH(-) and Cl(-) ions: K(B(2)OH), K(B(2)Cl). The proposed model has been proved to be correct by comparing the resulting theoretic charge variation curves of the PC-ST liposomal membrane with the experimental data.     

Selective Fe2+-catalyzed oxidative cleavage of gastric H+,K+-ATPase: implications for the energy transduction mechanism of P-type cation pumps

In the presence of ascorbate/H(2)O(2), Fe(2+) ions or the ATP-Fe(2+) complex catalyze selective cleavage of the alpha subunit of gastric H(+),K(+)-ATPase. The electrophoretic mobilities of the fragments and dependence of the cleavage patterns on E(1) and E(2) conformational states are essentially identical to those described previously for renal Na(+),K(+)-ATPase. The cleavage pattern of H(+),K(+)-ATPase by Fe(2+) ions is consistent with the existence of two Fe(2+) sites: site 1 within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and M1. The change in the pattern of cleavage catalyzed by Fe(2+) or the ATP-Fe(2+) complex induced by different ligands provides evidence for large conformational movements of the N, P, and A cytoplasmic domains of the enzyme. The results are consistent with the Ca(2+)-ATPase crystal structure (Protein Data Bank identification code; Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647-655), an E(1)Ca(2+) conformation, and a theoretical model of Ca(2+)-ATPase in an E(2) conformation (Protein Data Bank identification code ). Thus, it can be presumed that the movements of N, P, and A cytoplasmic domains, associated with the E(1) <--> E(2) transitions, are similar in all P-type ATPases. Fe(2+)-catalyzed cleavage patterns also reveal sequences involved in phosphate, Mg(2+), and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E(1) <--> E(2) transitions, and subconformations induced by H(+),K(+)-ATPase-specific ligands such as SCH28080.     

3'-azido-3'-deoxythymidine. An unusual nucleoside analogue that permeates the membrane of human erythrocytes and lymphocytes by nonfacilitated diffusion

The demonstrated in vitro and in vivo activity of 3'-azido-3'-deoxythymidine (N3dThd) against the infectivity and the cytopathic effect of human immunodeficiency virus has prompted an investigation of the mechanism by which this nucleoside analogue permeates the cell membrane. As with the transport of thymidine, the influx of N3dThd into human erythrocytes and lymphocytes was nonconcentrative during short incubation times (less than 5 min) which did not allow significant metabolism of this nucleoside. However, in contrast with thymidine transport, the initial velocity of N3dThd influx was strictly a linear function of nucleoside concentration (0.5-10 mM), without evidence of saturability; insensitive to micromolar concentrations of potent inhibitors of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep); insensitive to a 1000-fold excess of other nucleosides (thymidine, uridine, 2-chloroadenosine); and relatively insensitive to temperature, with Q10 values (37-27 degrees C) of 1.4 and 2.7 for N3dThd and thymidine, respectively, determined in erythrocytes. Although the above results indicate that N3dThd permeates the cell membrane chiefly by nonfacilitated diffusion and not via the nucleoside transporter, millimolar concentrations of this nucleoside analogue were observed to inhibit both zero-trans influx of thymidine and efflux of thymidine from [3H]thymidine-loaded erythrocytes. The partition coefficients (1-octanol:0.1 M sodium phosphate, pH 7.0) of N3dThd and thymidine were determined to be 1.26 and 0.064, respectively. The unusual ability of N3dThd to diffuse across cell membranes independently of the nucleoside transport system may be attributed to the considerable lipophilicity imparted to this molecule by the replacement of the 3'-hydroxyl group of thymidine with an azido moiety.     

Time-dependent effect of leptin on renal Na+,K+-ATPase activity

Leptin, secreted by adipose tissue, is involved in the pathogenesis of arterial hypertension, however, the mechanisms through which leptin increases blood pressure are incompletely elucidated. We investigated the effect of leptin, administered for different time periods, on renal Na(+),K(+)-ATPase activity in the rat. Leptin was infused under anesthesia into the abdominal aorta proximally to the renal arteries for 0.5-3 h. Leptin administered at doses of 1 and 10 microg/min per kg for 30 min decreased the Na(+),K(+)-ATPase activity in the renal medulla. This effect disappeared when the hormone was infused for > or =1 h. Leptin infused for 3 h increased the Na(+),K(+)-ATPase activity in the renal cortex and medulla. The stimulatory effect was abolished by a specific inhibitor of Janus kinases (JAKs), tyrphostin AG490, as well as by an NAD(P)H oxidase inhibitor, apocynin. Leptin increased urinary excretion of hydrogen peroxide (H(2)O(2)) between 2 and 3 h of infusion. The effect of leptin on renal Na(+),K(+)-ATPase and urinary H(2)O(2) was augmented by a superoxide dismutase mimetic, tempol, and was abolished by catalase. In addition, infusion of H(2)O(2) for 30 min increased the Na(+),K(+)-ATPase activity. Inhibitors of extracellular signal regulated kinases (ERKs), PD98059 or U0126, prevented Na(+),K(+)-ATPase stimulation by leptin and H(2)O(2). These data indicate that leptin, by acting directly within the kidney, has a delayed stimulatory effect on Na(+),K(+)-ATPase, mediated by JAKs, H(2)O(2) and ERKs. This mechanism may contribute to the abnormal renal Na(+) handling in diseases associated with chronic hyperleptinemia such as diabetes and obesity.     

Dissociation constants of protonated methionine species in NaCl media

The sulfur-containing amino acid, methionine, has a role in the physiological environment because of its strong interactions with metals. To understand these interactions of metals with methionine, one needs reliable dissociation constants for the protonated methionine species (NH(3)(+)CH(CH(2)CH(2)SCH(3))COOH; H(2)B(+)). The values of stoichiometric dissociation constants, pK(i)*, for protonated methionine species (H(2)B(+) if H(+)+HB, K(1); HB if H(+)+B(-), K(2)) were determined from potentiometric measurements in NaCl solutions as a function of ionic strength, 0.25-6.0 mol (kg H(2)O)(-1) and temperature (5-45 degrees C). The results were extrapolated to pure water using the Pitzer equations to estimate the activity of H(+), H(2)B(+), HB and B(-) as a function of ionic strength and temperature. The resulting thermodynamic values of K(1) and K(2) were fit to the equations (T/K): ln K(1)=69.0013-3496.58/(T/K)-10.9153 ln (T/K); ln K(2)=116.4162-10638.02/(T/K)-18.0553 ln (T/K) with standard errors of 0.003 and 0.033, respectively, for ln K(1)* and ln K(2)*. Pitzer interaction parameters (lambda(HB-Na) and zeta(HB-Na-Cl)) for the neutral HB were determined from literature data. The Pitzer parameters (beta(0)(H(2)BCl), beta(1)(H(2)BCl) and C(phi)(H(2)BCl)) for the interactions of H(2)B(+) with Cl(-) and Na(+) with and B(2-) (beta(0)(NaB), beta(1)(NaB) and C(phi)(NaB)) were also determined. These coefficients can be used to make reasonable estimates of the activity coefficients of methionine species and the pK(i)(*) for the dissociation of methionine in physiological solutions, composed mostly of NaCl over a wide range of temperature and ionic strength.     

An ONIOM investigation on anion recognition of alkali–metal complexes with diurea calix[4]arene receptor

The ONIOM(B3LYP/6-31G(d):AM1) optimized structures of complexes of diurea calix[4]arene receptor (L) with alkali metals Li(+), Na(+) and K(+) and their complexes with halide ions F(-), Cl(-), Br(-), oxygen-containing anions HCO(3)(-), HSO(4)(-) and CH(3)COO(-) ions were obtained. Binding energies and thermodynamic properties of complex receptors LiL(+), NaL(+) and KL(+) with these anions were determined. The binding stabilities according to binding energies of LiL(+), NaL(+) and KL(+) associated with anions computed either at the ZPVE-corrected ONIOM(B3LYP/6-31G(d):AM1) or BSSE-corrected B3LYP/6-31 + G(d,p)//ONIOM(B3LYP/6-31G(d):AM1) are in the same order: F(-) > CH(3)COO(-) ≈ HCO(3)(-) > Br(-) ≈ HSO(4)(-) ≈ Cl(-). All the receptors LiL(+), NaL(+) and KL(+) were found to be selective toward fluoride ion.     

Enzymes of pyrimidine deoxyribonucleotide metabolism in Mycoplasma mycoides subsp. mycoides.

Cell-free extracts of Mycoplasma mycoides subsp. mycoides were assayed for enzymes associated with the salvage synthesis of pyrimidine deoxyribonucleotides. They possessed kinases for deoxycytidine, (d)CMP, thymidine (deoxyuridine), dTMP, and nucleoside diphosphates; dCTPase and dUTPase; dCMP deaminase; thymidine (deoxyuridine) phosphorylase; and dUMP (dTMP) phosphatase. The existence of these enzymic activities together with ribonucleoside diphosphate reductase explains the capacity of cytidine to provide M. mycoides with deoxyribose for the synthesis of thymidine nucleotides from thymine.