Comparative study of the reactability of phosphoric acid residues in tRNA(Ser) and tRNA(Leu) from Thermus thermophilus

The reactivity of phosphates in the Thermus thermophilus tRNA(Ser) (GCU) and tRNA(Leu) (CAG) was studied using the ethylnitrosourea modification. It was shown that phosphates of nucleotides 58-60 (T loop), 20-22 (D loop), and 48 (at the junction of the variable and T stems) were poorly modified in both tRNAs. The most pronounced differences in the reactivity were observed for phosphates at the junctions of the variable stem with T-stem (47q, 49) and anticodon stem (45). This indicates differences in orientations of the long variable arm relative to the backbone in the tRNAs studied.     

Yeast tRNAAsp tertiary structure in solution and areas of interaction of the tRNA with aspartyl-tRNA synthetase. A comparative study of the yeast phenylalanine system by phosphate alkylation experiments with ethylnitrosourea

Ethylnitrosourea is an alkylating reagent preferentially modifying phosphate groups in nucleic acids. It was used to monitor the tertiary structure, in solution, of yeast tRNAAsp and to determine those phosphate groups in contact with the cognate aspartyl-tRNA synthetase. Experiments involve 3' or 5'-end-labelled tRNA molecules, low yield modification of the free or complexed nucleic acid and specific splitting at the modified phosphate groups. The resulting end-labelled oligonucleotides are resolved on polyacrylamide sequencing gels and data analysed by autoradiography and densitometry. Experiments were conducted in parallel on yeast tRNAAsp and on tRNAPhe. In that way it was possible to compare the solution structure of two elongator tRNAs and to interpret the modification data using the known crystal structures of both tRNAs. Mapping of the phosphates in free tRNAAsp and tRNAPhe allowed the detection of differential reactivities for phosphates 8, 18, 19, 20, 22, 23, 24 and 49: phosphates 18, 19, 23, 24 and 49 are more reactive in tRNAAsp, while phosphates 8, 20 and 22 are more reactive in tRNAPhe. All other phosphates display similar reactivities in both tRNAs, in particular phosphate 60 in the T-loop, which is strongly protected. Most of these data are explained by the crystal structures of the tRNAs. Thermal transitions in tRNAAsp could be followed by chemical modifications of phosphates. Results indicate that the D-arm is more flexible than the T-loop. The phosphates in yeast tRNAAsp in contact with aspartyl-tRNA synthetase are essentially contained in three continuous stretches, including those at the corner of the amino acid accepting and D-arm, at the 5' side of the acceptor stem and in the variable loop. When represented in the three-dimensional structure of the tRNAAsp, it clearly appears that one side of the L-shaped tRNA molecule, that comprising the variable loop, is in contact with aspartyl-tRNA synthetase. In yeast tRNAPhe interacting with phenylalanyl-tRNA synthetase, the distribution of protected phosphates is different, although phosphates in the anticodon stem and variable loop are involved in both systems. With tRNAPhe, the data cannot be accommodated by the interaction model found for tRNAAsp, but they are consistent with the diagonal side model proposed by Rich & Schimmel (1977). The existence of different interaction schemes between tRNAs and aminoacyl-tRNA synthetases, correlated with the oligomeric structure of the enzyme, is proposed.     

Studies on avian erythrocyte metabolism. Effect of organic phosphates on oxygen affinity of embryonic and adult-type hemoglobins of the chick embryo.

The effects of 2,3 diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), and inositol hexaphosphate (IHP) on the oxygen affinity of whole “stripped” hemoglobin (WSH), hemoglobin H (Hb-H), hemoglobin A (Hb-A) and hemoglobin D (Hb-D) isolated from 18-day chick embryo blood have been determined. The effect of the three organic phosphates upon the oxygen dissociation curves is similar and the following order of decreasing oxygen affinity of the organic phosphates was observed for each hemoglobin: 2,3-DPG < ATP < IHP. 2,3-DPG appears to have a slightly greater effect upon the P₅₀ of Hb-H than upon that of either of the two adult-type hemoglobins. However, this effect seems insufficient to suggest a preferential interaction of 2,3-DPG with Hb-H which would account for either the large amounts of 2,3-DPG in the erythrocytes of embryos or the higher oxygen affinity of the whole blood. The effects of the organic phosphates upon the Hill constant of the purified hemoglobins are variable. It is concluded that since the distribution of hemoglobins H, A, and D in the erythrocytes during the developmental period from 18-day embryos to 6-day chicks remains fairly constant, the previously described progressive decrease in oxygen affinity of the whole blood during this period results from changes in the total amount and distribution of the intraerythrocytic organic phosphates.²     

Study of the Ria Formosa ecosystem: benthic nutrient remineralization and tidal variability of nutrients in the water

Exchange of nutrients across the sediment-water interfaces of Ria Formosa was studied in the laboratory. In the field, water samples were collected (i) fortnightly, at low and high tide over one year, and (ii) semi-diurnally, over three neap-spring tidal cycles in winter. Results from both laboratory experiments and field collection were quite variable. Higher liberation rates of silicates, phosphates and ammonium occurred in chambers whose bottom was formed by mixtures of mud and sand covered by vegetation with clams. The bottom took up nitrates from the overlying water. Nutrient variations in the lagoon water were controlled both seasonally and tidally. Higher concentrations of silicates were found at low tide independently of the season; phosphates in spring/summer at low tide; nitrates in winter/spring at high tide. Tidal flushing appears to be an important removal-mechanism in this lagoon.     

Epidermal growth factor stimulates phosphatidylinositol turnover for ten hours in A431 cells without activation of protein kinase C

Epidermal Growth Factor stimulated phosphatidylinositol turnover in A431 cells for up to ten hours. There was an increase in phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate and phosphatidic acid at all time points. The effects on the inositol phosphates were variable. Despite the activation of the phosphatidylinositol cycle, we were unable to demonstrate activation of protein kinase C.     

NBD-labeled derivatives of the immunomodulatory drug FTY720 as tools for metabolism and mode of action studies

Fluorescently labeled chiral analogs of the immunomodulatory drug FTY720 and its corresponding phosphates with variable aliphatic spacers between the aromatic ring and the NBD label have been synthesized. Determining the influence of the spacer on the in vitro phosphorylation rate by SPHK1 and 2 resulted in the identification of NBD-(R)-AAL 1c,d which are phosphorylated with an efficiency comparable to that of the unlabeled FTY720 analog (R)-AAL. Furthermore, the NBD-(R)-AAL phosphates 10c,d were proven to be a functional S1P receptor agonist.     

Synthesis and biological activity investigation of azole and quinone hybridized phosphonates

Phosphonates, azoles and quinones are pharmacophores found in bioactive compounds. A series of phosphonates conjugated to azoles and quinones with variable carbon chain lengths were synthesized in 3–4 steps with good yield. Antifungal assay of these compounds showed that ethyl protected phosphates have excellent inhibitory activity against phytopathogenic fungus Fusarium graminearum, and the free-base phosphates have good activity against human pathogenic fungi Aspergillus flavus and Candida albicans. Structure- activity relationship (SAR) studies showed activity increases with longer carbon chain length between phosphonate and anthraquinone analogs consisting of azole and quinone moieties. These newly synthesized compounds also have mild antibacterial activities to Gram positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Cytotoxicity analysis of these compounds against HeLa cells reveals that the phosphoric acid analogs are less toxic compared to ethyl protected phosphonates. Three leads compounds have been identified with prominent antifungal activity and low cytotoxicity.     

In vivo 31P NMR spectroscopy of energy rich phosphates in the brain of the hyperammonemic rat

Hyperammonemia is a major contributing factor to the neurological abnormalities observed in hepatic encephalopathy and in congenital defects of ammonia detoxication. In rats variable changes in labile energy rich phosphates in the brain have been observed in hyperammonemia using biochemical methods. Using 31P-NMR spectroscopy however no significant changes of the relative concentrations of the energy rich phosphates alpha, beta and gamma-ATP, phosphocreatine, inorganic phosphate and the pH were found in the fronto parietal cortex of the urease treated hyperammonemic rat. Alterations in the metabolites of these compounds do not appear to be a major pathomechanism of ammonia toxicity in this brain area.     

How does the mood stabilizer lithium bind ATP,the energy currency of the cell: Insights from solid-state NMR

BackgroundLithium, in the form of a salt, is a mood stabilizer and a leading drug for the treatment of bipolar disorder. It has a very narrow therapeutic range and a variety of side effects. Lithium can replace magnesium and other cations in enzymes and small molecules, among them ATP, thereby affecting and inhibiting many biochemical pathways. The form of binding of lithium ions to ATP is not known.MethodsHere we extract the binding environment of lithium in solid ATP using a multi-nuclear multi-dimensional solid-state NMR approach.ResultsWe determine that the coordination sphere of lithium includes, at a distance of 3.0(±0.4) Å, three phosphates; the two phosphates closest to the ribose ring from one ATP molecule, and the middle phosphate from another ATP molecule. A water molecule most probably completes the fourth coordination. Despite the use of excess lithium in the preparations, sodium ions still remain bound to the sample, at distances of 4.3–5.5 Å from Li, and coordinate the first phosphate and two terminal phosphates.ConclusionsSolid-state NMR enables to unravel the exact coordination of lithium in ATP showing binding to three phosphates from two molecules, none of which are the terminal gamma phosphate.General significanceThe methods we use are applicable to study lithium bound to a variety of ATP-bound enzymes, or to other cellular targets of lithium, consequently suggesting a molecular basis for its mode of action.     

A modification of the Roe procedure for determination of fructose in tissues with increased specificity

A new modification of the procedure of Roe for determination of fructose concentrations in tissues is described. The modification shows no reaction with glucose or glucose phosphates. Tissues are homogenized in dilute perchloric acid, filtered, and reacted with alcoholic resorcinol and 30% HCl for 1 hr at 80°C. As little as 0.33 μg/ml of fructose may be reliably determined. Fructose phosphates react in this test but to a lesser extent than pure fructose.     

Reactivation of the alternative oxidase of Yarrowia lipolytica by nucleoside monophosphates

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of intact mitochondria. The incubation of mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and the detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated with AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order: AMP = GMP > GDP > GTP > MP > IMP. The apparent K(m) values for AMP in reactivation of the alternative oxidase of submitochondrial particles or mitochondria treated with Triton X-100 and incubated at 25 degrees C were calculated. Physiological aspects of activation of the alternative oxidase are discussed in connection with the impairment of electron transfer through the cytochrome pathway.     

Mechanism of Action of Showdomycin

The effect of showdomycin on the syntheses of deoxyribonucleotides from various pyrimidine and purine derivatives was studied in cell-free systems from E. coli.

The formations of deoxycytidine phosphates, deoxyuridine phosphates, deoxyguanosine phosphates and deoxyadenosine phosphates from the corresponding ribonucleoside diphosphates were all inhibited by low concentrations of showdomycin. The formation of deoxythymidine phosphates from dUMP was also very susceptible to the antibiotic. These inhibitory actions of showdomycin could be reversed by a sulfhydryl compound (mercaptoethanol) but not by nucleosides, in contrast to a previous finding that the inhibitory action of this antibiotic on the cell growth was reversed by compounds belonging to both of these groups.

N-Ethylmaleimide (NEM), a thiol reagent which has a structure related to the aglycone moiety of showdomycin, was also found to be a potent inhibitor of both the reduction of CDP and the methylation of dUMP as showdomycin. A mercurial thiol reagent, p-chloromercuribenzoic acid (PCMB), however, was found to be inactive against the methylation of dUMP although the salvage synthesis of dUMP was inhibited by low concentrations of this reagent.

The formations of deoxythymidine phosphates and of deoxyuridine phosphates from their respective pyrimidine bases and a deoxyribosyl donor were quite resistant to showdomycin.     

O-Alkylation of Dialkyl Phosphates and O-Alkyl Phosphonates via the Stannyl Intermediates

Abstract

A preparation of trialkyl phosphates and O, O dialkyl phosphonates by means of alkylation of the corresponding tributylstannyl dialkyl phosphates and tributylstannyl O-alkyl phosphonates was described.     

Effects of Kainic Acid on High-Energy Metabolites in the Mouse Striatum

Abstract: Intrastriatal injection of either kainic acid (0.35 μg) or ibotenic acid (7.0 μg) in the mouse causes a profound and selective degeneration of striatal neurons accompanied by a secondary astrocytic response. The kainate injection (0.35 μg) resulted in significant decrements in the striatal levels of phosphocreatine and ATP by 30 min, a progressive reduction in adenosine phosphates between 30 min and 48 h, and a decrease in energy charge; whereas lactate levels increased by 44% at 2 h, glucose levels fell by 56%. Two hours after intrastriatal injection of ibotenic acid (7.0 μg) similar alternations in striatal high-energy phosphates and glucose disposition were found. Prior decortication protected against the neurotoxic effects of kainate in the mouse striatum and prevented the alterations in high-energy phosphates at 2 h although lactate levels increased by 212%. These findings in vivo are consistent with the hypothesis that the neurotoxic effects of acidic excitatory amino acids involve a profound activation of energy consumption by affected neurons.     

A comparative study of metabolite levels in plant leaf material in the dark

Metabolite levels have been compared in the dark and during photosynthesis in leaves and protoplasts from spinach, pea, wheat and barley. In protoplasts the subcellular distribution was also studied. The levels of triose phosphates and sugar bisphosphates were high in the light and low in the dark. The hexose phosphates and 3-phosphoglycerate levels in the dark were very variable depending on the plant material. In most conditions, hexose phosphates and triose phosphates were mainly in the extrachloroplast compartment, while 3-phosphoglycerate and the sugar bisphosphates were mainly in the chloroplast compartment. Leaves always had a very low triose phosphate: 3-phosphoglycerate ratio in the dark, but in protoplasts this ratio was higher. Detailed studies with spinach showed that metabolite levels were very dependent on the availability of carbohydrate in the leaf, particularly starch. Starch mobilisation is not controlled just by the availability of inorganic phosphate and accumulation of phosphorylated intermediates. Hydrolysis of starch may provide precursors for sucrose synthesis while phosphorolysis leads to provision of substrates for respiration. Starch breakdown generates high enough levels of hexose phosphate to support substantial rates of sucrose synthesis in the dark. Respiration is not greatly increased when metabolite levels are high during starch mobilisation. Higher levels of metabolites shorten the length of the induction phase of photosynthesis.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - Fru2,6bisP fructose-2,6-bisphosphate - NMR nuclear magnetic resonance - PGA 3-phosphoglyceric acid - Pi inorganic phosphate - RuBP ribulose-1,5-bisphosphate - UDPGlc uridine-5-diphosphate glucose     

Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers.

Since the original work by Sweeley et al. (1) the silylation and subsequent gas-liquid chromatography of simple sugars has been well documented (2–5). The silylation of their biochemically important derivatives, notably sugar phosphates, has proven more difficult. Wells et al. (6) premethylated the phosphate with diazomethane in methanol before silylating the sugar group with the usual reagents [pyridine, hexamethyldisilazane (HMDS), and trimethylchlorosilane (TMCS)], Hashizume and Sasaki (7) refluxed the sugar phosphates in the reaction mixture for 1 hr to effect complete silylation, while Eisenberg and Bolden (8) achieved similar results by heating in a sealed vial at 100°C for 10 min. The latter researchers noted that glucose 1-phosphate tended to decompose under their conditions to give one major and two minor peaks. Horning et al. (9) and, later, Sherman et al. (10) used bis(trimethylsilyl)acetamide (BSA) to silylate sugar phosphates, but Pierce (11) has noted that BSA tends to give inconsistent silylation of simple sugars and we have also noted this in our work.In 1969, Ellis (12) reported on the use of a silylation system using dimethylsuifoxide (DMSO) or dimethylformamide (DMF) as the reaction solvent rather than pyridine. The silylated derivatives are not soluble in either of these solvents and they form a second, upper liquid layer (in 10 min in the case of DMSO, 18 hr for DMF). The silylated sample is thus concentrated and this avoids the necessity of removing the solvent [with its attendant problems (10)] after silylation.This paper reports a modification and extension of the silylation system described by Ellis which allows rapid, quantitative silylation of sugars and sugar phosphates at room temperature, i.e., the addition of cyclohexane to the silylation mixture to promote the silylation of sugar phosphates and other biologically important acids. Also reported is the first use of SE-52 stationary liquid phase for the glc of sugar phosphates.     

Isolation of soluble metabolites of the phosphatidylinositol cycle from Samanea saman

An improved protocol for the separation of inositol phosphates by high performance liquid chromatography was used to resolve inositol phosphates from pulvini (motor organs) of the legume, Samanea saman. The pulvini contained inositol phosphate, inositol bisphosphate, and inositol trisphosphate isomers which co-migrated with those of mammalian red blood cells, and one or more other inositol metabolites which, to our knowledge, have not been previously noted in preparations of inositol phosphates. The finding of inositol phosphates in Samanea which comigrate with mammalian inositol phosphates supports the possibility that the phosphatidylinositol cycle may function in signal transduction in plants as well as in animals.Abbreviations HPLC high performance liquid chromatography - PI phosphatidylinositol     

Continuous colorimetric monitoring of the fructose bisphosphate aldolase reaction

A simple method for continuous spectrophotometric assay of fructose-1,6-bisphosphate aldolase is described. The method is based on the reactivity of the product triose phosphates with cyanide to form compounds capable of the reduction of cytochrome c. At the concentrations employed, cyanide, acting catalytically, reacts with the enediol tautomer of the triose phosphates to generate the reductants, which reduce cytochrome c causing increased absorbance at 550 nm. The rate of increase in the rate of appearance of 550-nm absorbance is directly proportional to the concentration of aldolase present. The procedure is particularly useful for Class I aldolases, since the assay must be run under mildly alkaline conditions.     

Analysis of the interaction of organic phosphates with hemoglobin.

The interaction of organic phosphates with hemoglobin is studied by use of a simple thermodynamic approach. A model-independent analysis is employed to evaluate the accuracy of Adair constants determined in the presence of 2,3-diphosphoglycerate (DPG). The change of oxygen affinity in the presence of phosphates is related to the macroscopic phosphate binding constants of oxy- and deoxyhemoglobin and used to extract such binding constants from oxygen equilibrium measurements. The change of the Bohr effect in the presence of phosphates and the competitive binding of carbon dioxide and DPG are treated quantitatively. The binding of organic phosphates is incorporated into an allosteric model, in which the effect of phosphate on both tertiary and quaternary structure changes is included. By use of this model, the factors which can be responsible for the increased functional heterogeneity of alpha and beta chains in the presence of phosphates are clarified.     

Tertiary structure of Escherichia coli tRNA(3Thr) in solution and interaction of this tRNA with the cognate threonyl-tRNA synthetase

The solution structure of Escherichia coli tRNA(3Thr) (anticodon GGU) and the residues of this tRNA in contact with the alpha 2 dimeric threonyl-tRNA synthetase were studied by chemical and enzymatic footprinting experiments. Alkylation of phosphodiester bonds by ethylnitrosourea and of N-7 positions in guanosines and N-3 positions in cytidines by dimethyl sulphate as well as carbethoxylation of N-7 positions in adenosines by diethyl pyrocarbonate were conducted on different conformers of tRNA(3Thr). The enzymatic structural probes were nuclease S1 and the cobra venom ribonuclease. Results will be compared to those of three other tRNAs, tRNA(Asp), tRNA(Phe) and tRNA(Trp), already mapped with these probes. The reactivity of phosphates towards ethylnitrosourea of the unfolded tRNA was compared to that of the native molecule. The alkylation pattern of tRNA(3Thr) shows some similarities to that of yeast tRNA(Phe) and mammalian tRNA(Trp), especially in the D-arm (positions 19 and 24) and with tRNA(Trp), at position 50, the junction between the variable region and the T-stem. In the T-loop, tRNA(3Thr), similarly to the three other tRNAs, shows protections against alkylation at phosphates 59 and 60. However, tRNA(3Thr) is unique as far as very strong protections are also found for phosphates 55 to 58 in the T-loop. Compared with yeast tRNA(Asp), the main differences in reactivity concern phosphates 19, 24 and 50. Mapping of bases with dimethyl sulphate and diethyl pyrocarbonate reveal conformational similarities with yeast tRNA(Phe). A striking conformational feature of tRNA(3Thr) is found in the 3'-side of its anticodon stem, where G40, surrounded by two G residues, is alkylated under native conditions, in contrast to other G residues in stem regions of tRNAs which are unreactive when sandwiched between two purines. This data is indicative of a perturbed helical conformation in the anticodon stem at the level of the 30-40 base pairs. Footprinting experiments, with chemical and enzymatic probes, on the tRNA complexed with its cognate threonyl-tRNA synthetase indicate significant protections in the anticodon stem and loop region, in the extra-loop, and in the amino acid accepting region. The involvement of the anticodon of tRNA(3Thr) in the recognition process with threonyl-tRNA synthetase was demonstrated by nuclease S1 mapping and by the protection of G34 and G35 against alkylation by dimethyl sulphate. These data are discussed in the light of the tRNA/synthetase recognition problem and of the structural and functional properties of the tRNA-like structure present in the operator region of the thrS mRNA.