Monophosphate Compounds Research Articles

AC conductivity and dielectric behavior of low-temperature phase of α-AgCuPO4

The copper monophosphate compound, α-AgCuPO4, was prepared by the conventional solid-state reaction. The purity of the compound was checked by X-ray diffraction technique (XRD). Detailed dielectric and electrical properties of the compound were analyzed as a function of frequency (209 Hz–1 MHz) and temperature (343–514 K). The AC conductivity has been fitted and studied using Jonscher’s equation, whose exponent n varied with the temperature, showing that the overlapping large polaron tunneling model (OLPT) is the appropriate model to describe the electrical conduction mechanism.

Phosphate Chemical Probes Designed for Location Specific Inhibition of Intracellular Carbonic Anhydrases.

Chemical probes are small molecules designed to bind to a specific protein and disrupt the proteins function. Although many inhibitors are reported for human carbonic anhydrase (CA) enzymes, few may be considered useful as chemical probes as they exhibit broad action against the 12 catalytically active CA isozymes. In addition, most do not possess an appropriate physicochemical profile to discriminate intracellular CA activity from either global or extracellular CA activity. We report herein the synthesis of three monophosphate CA proinhibitors (compounds 2, 3, and 5) that are derived from cyclosaligenyl (cycloSal) phosphate and S-acyl-2-thioethyl (SATE) phosphate as protecting groups. The proinhibitors are designed as neutral, membrane-permeable compounds that once inside the cell may be hydrolyzed by pH-driven or enzymatic-driven mechanisms to release a negatively charged monophosphate. The resulting monophosphate compound is trapped intracellularly and available for locality specific inhibition of intracellular CAs.

Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized in mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.7.1.21)-like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. The results obtained in the present study suggest a crucial role for the salvage of thymidine during early plant development.

A multisubstrate deoxyribonucleoside kinase from plants

Deoxyribonucleoside kinases catalyze the rate limiting step during the salvage of deoxyribonucleosides and convert them into the corresponding monophosphate compounds. We have identified and characterized a unique multisubstrate deoxyribonucleoside kinase from plants. The phylogenetic relationship and biochemical properties suggest that this deoxyribonucleoside kinase represents a living fossil resembling the progenitor of the modern animal deoxycytidine, deoxyguanosine and thymidine 2 kinases. The broad substrate specificity makes this enzyme an interesting candidate to be evaluated as a suicide gene in anti-cancer therapy.

Sorption of Uranium(VI) onto Lanthanum Phosphate Surfaces

The speciation of hexavalent uranium at the solid−solution interface was investigated. To experimentally identify the sorption equilibria, we characterized the structure of the surface complex formed by binding between uranyl ions and surface groups of solid matrixes. To understand the sorption mechanisms at a molecular level, we performed optical and X-ray photoelectron spectroscopies and X-ray absorption spectroscopy on uranyl ion loaded phosphate solids (LaPO4 and La(PO3)3). Two lanthanum phosphates were synthesized. The samples were contacted with aqueous uranyl solutions of pH values ranging from 1.0 to 4.0. Whatever the conditions, uranium surface coverage was always lower than 30% of the monolayer as measured by the proton-induced X-ray emission technique. The U 4f X-ray photoelectron spectra and the lifetime values of uranyl ions sorbed on the lanthanum monophosphate compound clearly evidence that this solid exhibits two different types of sorption sites, as well as lanthanum polytrioxophosphate. The nature of the site which interacts with uranyl ions also depends on the pH value for both solids. Moreover, the interaction of the uranyl ions and the phosphate solids in a nitrate medium leads to two different sorbed species: free aquo UO22+ ions and UO2(NO3)+ ions. The X-ray absorption spectroscopy performed on the sorbed samples gives evidence of the presence of an inner-sphere mononuclear polydentate surface complex.

Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP.

RNA transcripts containing the hammerhead ribozyme have been engineered to self-destruct in the presence of specific nucleoside 3',5'-cyclic monophosphate compounds. These RNA molecular switches were created by a new combinatorial strategy termed 'allosteric selection,' which favors the emergence of ribozymes that rapidly self-cleave only when incubated with their corresponding effector compounds. Representative RNAs exhibit 5,000-fold activation upon cGMP or cAMP addition, display precise molecular recognition characteristics, and operate with catalytic rates that match those exhibited by unaltered ribozymes. These findings demonstrate that a vast number of ligand-responsive ribozymes with dynamic structural characteristics can be generated in a massively parallel fashion. Moreover, optimized allosteric ribozymes could serve as highly selective sensors of chemical agents or as unique genetic control elements for the programmed destruction of cellular RNAs.

Partial characterization of the dissolved organic phosphorus pool in the oligotrophic North Pacific Ocean

The combined application of two independent methods for phosphorus (P i determination in seawater, continuous‐flow UV photodecomposition and magnesium‐induced coprecipitation (MAGIC), was used to provide a partial characterization of the dissolved P pools at Sta. ALOHA (22°45′N, 158°W) in tl e oligotrophic subtropical North Pacific Ocean. Comprehensive laboratory analyses of the UV light‐induced photodecomposition of a variety of specific organic P compounds dissolved in seawater confirmed that submicromolar concentrations of monophosphate compounds could be analytically separated from nucleotide di‐ and triphosphates based upon a previously described temperature‐controlled low‐pressure, mercury vapor UV irradiation treatment. When combined with a recently described high‐precision P‐detection system, the total dissolved phosphorus (TDP) in seawater could be reproducibly subdivided into three chemically distinct pools: soluble reactive phosphorus (SRP; presumably dominated by orthophosphate), UVlabile phosphorus (PUV‐L; containing primarily monophosphate esters), and UV‐stable phosphorus (PUV‐S; containing primarily nucleotide di‐ and triphosphates, nucleic acids, and other compounds that are resistant to the UV treatment). Field application of these procedures to samples collected at Sta. ALOHA during the period September 1991 to March 1992 (HOT‐30 to HOT‐35) revealed the presence of all three operationally defined pools. In the upper portion of the water column (0–100 m) the TDP pool (29.26 ± 2.32 mmol P m−2) contained, on average, 23% SRP, 26% PUV‐S, and 51% With increasing depth, the concentration of P decreased while that of the PUV‐S pool increased; the PUVL,: PUV‐S ratios decreased from values of 2–5 in the upper water column to ≤ 1.0 at 200 m.

An exploration of the binding site of aldolase using alkyl glycolamido phosphoric esters and alkyl monoglycolate phosphoric esters

Alkyl glycolamido phosphoric esters (P-O-CH2-CO-NH-(CH2)n-CH3) and alkyl monoglycolate phosphoric esters (P-O-CH2-CO-O-(CH2)n-CH3), which are analogs of the aldolase substrate fructose-1-phosphate, were synthesized and use for probing the active site of rabbit muscle aldolase. The inhibition constants (Ki) were affected by the length of the alkyl groups of these compounds and a maximum value of Ki was observed between the number of methylene groups 2 and 4, depending on the type of compound. In the previous investigation, N-(omega-hydroxyalkyl)-glycolamido bisphosphoric esters (P-O-CH2-CO-NH-(CH2)n-O-P) and alkanediol monoglyclolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P) have a minimum Ki value between the number of methylene groups 1 and 4. The difference spectra of aldolase caused by binding of alkyl glycoamido phosphoric esters or alkyl monophosphates resembled that of their analogous bisphosphoric esters, but the intensity of absorbance was smaller than that of the bisphosphoric ester analogs. These results suggest that rabbit muscle aldolase has two binding sites for the phosphate groups on the entrance end of the active site cavity, the singly wound beta-barrel of the parallel alpha/beta class structure. The distance between the phosphate binding site Lys-107 in the beta-sheet structure (c) and Arg-148 in the beta-sheet structure (d) may possibly be expanded or contracted by the forms of the bending structure of the biphosphate compounds. Also, the change of distance between the beta-sheet structure (c) and (d) containing Trp-147, may have an effect on the environment of the tryptophan and cause a change of the absorbance of aldolase especially at 295-299 nm. On the other hand, the synthetic monophosphate compounds bind at only one of the two phosphate binding sites and have very little effect on the absorbance of Trp-147, in a similar manner as orthophosphate. The alkyl groups of monophosphate may be repelled by the ionic amino acid side chains, Asp-33, Lys-146, Glu-187 and/or Lys-229 in the middle of the active site cavity. However, the end of the long alkyl group of some monophosphates may possibly contact the hydrophobic bottom of the active site cavity without effect on the environment of Trp-147.

ＡＴＰによる魚類ミオシンの熱変性抑制の再検討

The mechanism involved in protective effect of ATP on thermal inactivation of myosin Ca-ATPase was re-examined. 1) ATP concentration required for full protection against thermal denaturation is myosin concentration dependent, and two concentrations were similar to each other. 2) The protection by ATP is more effective in the presence of Mg2+ than in the presence of Ca2+ or EDTA, suggesting the high affinity of ATP-Mg to the ATPase active site. 3) The protective effects of triphosphate compounds (ATP and AMPPNP) were distinguish-able from those of diphosphate compounds (ADP, PPi) in their concentration dependence and in the maximal protection achieved, and monophosphate compounds (AMP and IMP) were unable to protect myosin from inactivation at all.

The effect of human C-reactive protein on the cell-attachment activity of fibronectin and laminin

We have previously reported that purified human C-reactive protein (CRP) specifically binds to the cell-binding region of plasma fibronectin (Fn) in a Ca 2+-dependent reaction that is saturable at a molar ratio of CRP/Fn of approximately 9. In this study, the binding of CRP to Fn was found to interfere with the cell-attachment promoting activity of Fn. The inhibition of cell attachment was dependent on the concentration of the CRP and involved the phosphorylcholine (PC) binding site of CRP since inhibition was prevented by allowing the CRP to react with either PC (or closely related monophosphate compounds) or a mAb specific for the PC-binding site of CRP. Binding of CRP to laminin was also Ca 2+-dependent; however, this binding did not alter the cell-attachment promoting activity of laminin. CRP by itself does not mediate cell attachment. Since CRP is selectively deposited at sites of tissue damage along with plasma Fn and has the ability to bind to Fn and alter its cell-binding activity, CRP may modulate early events in tissue repair.

A potent nucleoside triphosphate hydrolase from the parasitic protozoan Toxoplasma gondii. Purification, some properties, and activation by thiol compounds.

A potent nucleoside triphosphate hydrolase (EC 3.6.1.3) with a number of unusual properties has been found in the parasitic protozoan (Toxoplasma gondii) and has been purified to homogeneity. The enzyme is localized in the cytosol and constitutes 3-4% of the total cytosolic protein. It has a molecular weight of 240,000-260,000 and contains four equivalent subunits of Mr = 63,000. Dithiol compounds such as dithiothreitol, dithioerythritol, or dimercaptopropanol were essential activators of the enzyme. Monothiol compounds had no effect. The specific activity of the purified enzyme was 2,500 mumol/min/mg at 37 degrees C under optimal conditions. Magnesium was the most effective activating metal ion, although manganese and calcium were also active. A higher excess of magnesium over total ATP was essential for maximal activity. Anions were found to inhibit the enzyme activity in an almost chaotropic order. The enzyme demonstrated a wide substrate specificity for both ribo- and deoxyribonucleoside triphosphate and hydrolyzed these nucleotides at almost the same rate. ADP was also a substrate and was hydrolyzed at a rate of 18% of that for ATP. Slight activity was seen with inorganic tri- and tetrapolyphosphates but not with monophosphate compounds. Km values for MgATP2- and MgADP- were 0.12 +/- 0.01 mM and 0.70 +/- 0.06 mM, respectively.

31P nuclear magnetic resonance studies on relaxation parameters and line broadening of intracellular metabolites of HeLa cells

The 40-MHz 31P nuclear magnetic resonance (nmr) spectrum of intact HeLa cells contains seven broad peaks with some detectable splittings. The linewidths were significantly broader than for those of cell-free systems such as cell extracts, indicating that the cellular environment is responsible for the unusual line broadening. Resolution of these peaks at 40 MHz is sufficient to make certain assignments and the relaxation parameters of some of the intracellular metabolites have been measured. The spin-lattice relaxation times ( T 1) ranged from 0.3 s for adenosine triphosphate (ATP) to about 3 s for inorganic phosphate ( P i ) and monophosphate compounds. Nuclear Overhauser enhancements (NOE) were induced by proton irradiation with the possible exception of ATP. The relaxation parameters were compared to those of cell-free compounds and in all cases T 1 and NOE were smaller for the intracellular metabolites. The relaxation parameters for ATP were affected the most. This behavior was mimicked with mixtures of cell-free metabolites containing paramagnetic ions. The larger change in both T 1 and NOE of intracellular ATP could be accounted for by selective binding of paramagnetic ions. This phenomenon also explains some of the line broadening in the cell spectrum especially that of ATP. The spin-spin relaxation times ( T 2) of P 1 and monophosphate compounds as measured by a pulse technique did not account for the observed linewidths. This is due to the presence of chemical shift envelopes arising from pH heterogeneity. All resonances were broader at 146 MHz because of the line broadening by paramagnetic ions and the presence of chemical shift envelopes. Other mechanisms of line broadening may also be significant. There was little difference in resolution of spectra at 40 and 146 MHz. Water proton linewidths and T 2 values were measured for HeLa cells and for some minced tissue preparations. The water linewidth in tissue samples was broader than that in the cell suspension. The large linewidths in tissues arise mainly from chemical shift envelopes caused by magnetic field nonuniformity in the tissue samples. There appears to be a small chemical shift envelope from magnetic nonuniformity in HeLa cells as well. The 1H results on envelopes were extrapolated to 31P studies on cells and tissues. Possible methods for reducing linewidths arising from the various proposed broadening mechanisms were discussed.