Cyclic condensed metaphosphates and linear polyphosphates in brown and red algae

The occurrence of linear condensed polyphosphates and cyclic condensed metaphosphates was studied by means of pulse-labeling with ³²P-orthophosphate (3–5 h) in a number of Phaeophyceae species: Pylaiella litoralis, Ilea fascia, Ectocarpus siliculosus and also Rhodophyceae species: Ceramium deslongchampsii, C. rubrum, Rhodomela confervoides, Porphyridium purpureum and P. aerugineum. Twodimensional cellulose thin layer chromatography revealed that in all species studied ³²P-radioactivity was generally present in all oligopolyphosphates containing 2 to 7 phosphate residues, in cyclic metaphosphates (tri-, tetra-, penta- and hexametaphosphates) and in high-molecular-weight condensed phosphates which remained at the starting point. Among the low-molecular-weight condensed inorganic phosphates the trimetaphosphate had a significantly higher specific activity than the other oligophosphates which were separated on the chromatography plates as measured by the direct scanning with a Geiger-Muller counter.The phosphate uptake strongly depends on the internal pool of reserve phosphates of the algae cells. The ³²P-orthophosphate incorporation of the cells is low and sluggish when growning in a synthetic medium or in sea water. Accordingly ³²P appeared preferentially in the low-molecular-weight fractions of condensed phosphates since the storage phosphates were not yet used. After previous incubation in a P-free culture medium of the algae the ³²P was rather rapidly incorporated and was found mostly in the highmolecular-weight condensed phosphates.During MAK-chromatography the high-molecular-weight fractions were eluted together with the nucleic acids (tRNA and DNA) while most of the low-molecular-weight fractions left the column immediately on elution.     

Use of phosphorus-32 labeled polyphosphoric acid for synthesis of labeled purine and pyrimidine 5'-ribonucleotides

Phosphorus-32 labeled polyphosphoric acid was used to phosphorylate the 2′,3′-O-isopropylidene derivaties of purine and pyrimidine ribonucleosides. The reaction mixture, which contained isopropylidene ribonucleoside 5′-phosphates, was subjected to selective hydrolysis, which removed the isopropylidene protecting group. The polyphosphate chains remained intact. Chromatography revealed the presence of ribonucleoside 5′-mono-, di-, tri-, tetra-, and higher phosphates. The distribution of radioactive label was established by liquid scintillation counting and enzyme assay. The triply labeled adenosine triphosphate prepared in this manner proved to be enzymically active and gave a positive firefly bioluminescence response.     

Prebiotic phosphorylation of nucleosides in formamide.

A possible prebiotic phosphorylation method has been investigated in which formamide served as the reaction medium. Nucleotides and nucleotide derivatives were formed when nucleosides were allowed to react with different orthophosphate, hydrogen phosphate or dihydrogen phosphate salts or with different condensed phosphate salts. The reaction products obtained from the phosphorylation of adenosine were 2'3' and 5'-AMPs, 2',5' and 3',5'-ADPs and 2',3'-cyclic AMP. The extent of phosphorylation in formamide exceeded 50% under favorable conditions after 15 days at 70 degrees. The acidic dihydrogen phosphates and condensed hydrogen phosphates proved to be the best phosphorylating agents. The presence of water in the medium decreased the yield of nucleotide derivatives, but some phosphorylation of adenosine was detected using dihydrogen phosphate in formamide containing water. The phosphorylation reactions were also observed for deoxynucleosides. Little decompression of the nucleosides was detected during the reaction time needed to form nucleotide derivatives. The facility with which phosphorylation takes place in formamide under very mild conditions may justify further studies both of prebiotic phosphorylation and synthetic phosphorylation using this solvent.     

Active species for Ce(IV)-induced hydrolysis of phosphodiester linkage in cAMP and DNA

The hydrolysis of cyclic adenosine 3',5'-monophosphate and 2'-deoxythymidylyl(3'-5')2'-deoxythymidine by Ce(NH4)2(NO3)6 was kinetically studied. The rate of hydrolysis was fairly proportional to the concentration of [Ce2(IV) (OH)4]4+ , showing that this is the catalytically active species. According to quantum-chemical calculation, the two Ce(IV) ions in this [Ce2(IV) (OH)4]4+ cluster are bridged by two OH residues. Upon the complex formation with H2 PO4- (a model compound for the phosphodiesters), these two Ce(IV) ions bind the two oxygen atoms of the substrate and enhance the electrophilicity of the phosphorus atom. The catalytic mechanism of Ce(IV)-induced hydrolysis of phosphodiesters has been proposed on the basis these results.     

Cooperation of Co(III) complex and cyclodextrin for prompt and regioselective hydrolysis of ribonucleoside 2',3'-cyclic monophosphates at pH 7.

2',3'-Cyclic phosphates of guanosine and adenosine (G greater than p and A greater than p), the simplest intermediates in RNA hydrolysis, are regioselectively and promptly hydrolyzed at pH 7, 30 degrees C by combination of [Co(tme)2(H2O)2]3+ complex and cyclodextrins: tme (1,1,2,2-tetramethylethylenediamine). Both the selectivity and the reaction rate are largely increased by cooperation of these two catalysts.     

Effects of condensed phosphates on plant growth and phosphorus uptake

Summary Four condensed phosphates with large and small molecules, including ring and chain structures, were equivalent to orthophosphate in terms of phosphorus uptake and the dry matter yield of ryegrass grown in two soils in pots. This equivalence was maintained at each of 6 cuts during two seasons.Increasing N rate greatly increased uptake of applied phosphorus but there was no differential effect with different sources of phosphorus, nor was there any interaction between source and application rate.Phosphorus was not leached at any time, indicating that the phosphates were rapidly adsorbed by the soils.Fairly rapid hydrolysis of all the condensed phosphates occurred in a soil of neutral pH but a slower hydrolysis seemed to occur in an acid soil.There was a good correlation between the phosphorus uptake by ryegrass and the additional orthophosphate released by an acid hydrolysis of soil extracts.     

Kinetic studies on degradation of nucleotides catalyzed by phosphodiesterase-phosphomonoesterase from Fusarium moniliforme

Degradation of the 2'-phosphates, 3'-phosphates, 5'-phosphates, 2':3'-cyclic phosphates, 3':5'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) of adenosine, guanosine, cytidine, and uridine catalyzed by Fusarium phosphodiesterase-phosphomonoesterase was followed by means of high performance liquid chromatography. All the nucleotides were susceptible to the enzyme to a greater or lesser degree, and the kinetic constants, Km and kcat, were determined at pH 5.3 and 37 degrees C. These constants were affected by both the nucleoside moiety and the position of the phosphate. Judged from kcat/Km, the 3'-phosphates, 2':3'-cyclic phosphates, and 5'-(p-nitrophenylphosphates) were good substrates, whereas the 2'-phosphates, 5'-phosphates, and 3':5'-cyclic phosphates were poor substrates except for adenosine 2'-phosphate, adenosine 5'-phosphate, and cytidine 5'-phosphate, which were hydrolyzed relatively easily. Among the phosphodiesters, the 2':3'-cyclic phosphates of adenosine, guanosine, and cytidine; and the 3':5'-cyclic phosphates of adenosine and cytidine were degraded into nucleoside and inorganic phosphate without release of intermediary phosphomonoester into the medium. Other phosphodiesters were degraded stepwise releasing definite intermediates.     

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase.

In the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) through formation of the E-LH2-AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7. The Km values for luciferin and ATP are 2-3 microM and 4 mM, respectively. The synthesis is strictly dependent upon luciferin and a divalent metal cation. Mg2+ can be substituted with Zn2+, Co2+ or Mn2+, which are about half as active as Mg2+, as well as with Ni2+, Cd2+ or Ca2+, which, at 5 mM concentration, are 12-20-fold less effective than Mg2+. ATP is the best substrate of the above reaction, but it can be substituted with adenosine 5'-tetraphosphate (p4A), dATP, and GTP, and thus the luciferase synthesizes the corresponding homo-dinucleoside polyphosphates:diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A), dideoxyadenosine 5',5"'-P1,P4-tetraphosphate (dAp4dA) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). In standard reaction mixtures containing ATP and a different nucleotide (p4A, dATP, adenosine 5'-[alpha,beta-methylene]-triphosphate, (Ap[CH2]pp), (S')-adenosine-5'-[alpha-thio]triphosphate [Sp)ATP[alpha S]) and GTP], luciferase synthesizes, in addition to Ap4A, the corresponding hetero-dinucleoside polyphosphates, Ap5A, adenosine 5',5"'-P1,P4-tetraphosphodeoxyadenosine (Ap4dA), diadenosine 5',5"'-P1,P4-[alpha,beta-methylene] tetraphosphate (Ap[CH2]pppA), (Sp-diadenosine 5',5"'-P1,P4-[alpha-thio]tetraphosphate [Sp)Ap4A[alpha S]) and adenosine-5',5"'-P1,P4-tetraphosphoguanosine (Ap4G), respectively. Adenine nucleotides, with at least a 3-phosphate chain and with an intact alpha-phosphate, are the preferred substrates for the formation of the enzyme-nucleotidyl complex. Nucleotides best accepting AMP from the E-LH2-AMP complex are those which contain at least a 3-phosphate chain and an intact terminal pyrophosphate moiety. ADP or other NDP are poor adenylate acceptors as very little diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) or adenosine-5',5"'-P1,P3-triphosphonucleosides (Ap3N) are formed. In the presence of NTP (excepting ATP), luciferase is able to split Ap4A, transferring the resulting adenylate to NTP, to form hetero-dinucleoside polyphosphates. In the presence of PPi, luciferase is also able to split Ap4A, yielding ATP. The cleavage of Ap4A in the presence of Pi or ADP takes place at a very low rate. The synthesis of dinucleoside polyphosphates, catalyzed by firefly luciferase, is compared with that catalyzed by aminoacyl-tRNA synthetases and Ap4A phosphorylase.     

Are polyphosphates or phosphate esters prebiotic reagents?

It is widely held that there was a phosphate compound in prebiotic chemistry that played the role of adenosine triphosphate and that the first living organisms had ribose-phosphate in the backbone of their genetic material. However, there are no known efficient prebiotic synthesis of high-energy phosphates or phosphate esters. We review the occurrence of phosphates in Nature, the efficiency of the volcanic synthesis of P₄O₁₀, the efficiency of polyphosphate synthesis by heating phosphate minerals under geological conditions, and the use of high-energy organic compounds such as cyanamide or hydrogen cyanide. These are shown to be inefficient processes especially when the hydrolysis of the polyphosphates is taken into account. For example, if a whole atmosphere of methane or carbon monoxide were converted to cyanide which somehow synthesized polyphosphates quantitatively, the polyphosphate concentration in the ocean would still have been insignificant. We also attempted to find more efficient high-energy polymerizing agents by spark discharge syntheses, but without success. There may still be undiscovered robust prebiotic syntheses of polyphosphates, or mechanisms for concentrating them, but we conclude that phosphate esters may not have been constituents of the first genetic material. Phosphoanhydrides are also unlikely as prebiotic energy sources. Correspondence to: S.L. Miller     

Self‐assembly and chiroptical properties in supramolecular complexes of adenosine phosphates and guanidinium‐bispyrene

Supramolecular systems that respond to the hydrolysis of adenosine phosphates (APs) are attractive for biosensing and to fabricate bioinspired self‐assembled materials. Here, we report on the formation of supramolecular complexes between an achiral guanidinium derivative bearing two pyrene moieties, with each of the three adenosine phosphates: AMP, ADP, and ATP. By combining results from circular dichroism spectroscopy and molecular modeling simulations, we explore the induced chirality, the dynamics of the complexes, and the interactions at play, which altogether provide insights into the supramolecular self‐assembly between APs and the guanidinium‐bispyrene. Finally, we identify the chiroptical signals of interest in mixtures of the guanidinium derivative with the three APs in different proportions. This study constitutes a basis to evolve toward a chiroptical detection of the hydrolysis of APs based on organic supramolecular probes.     

Prebiotic peptide-formation in the solid state

The reactions of glycine with inorganic polyphosphates in the solid state have been studied. The formation of peptides up to the decamer occurs at moderate temperatures(r.t.-100 degrees C) in the presence of imidazole and magnesium chloride. If adenosine 5' -monophosphate is added to the reaction mixture, 2'(3') -o-glycyl adenosine 5'-monophosphate is also obtained. These reactions could have occurred on the primitive earth.     

4-Coumarate:coenzyme A ligase has the catalytic capacity to synthesize and reuse various (di)adenosine polyphosphates

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono- and diadenosine polyphosphates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of adenosine 5'-tetraphosphate (p(4)A) and adenosine 5'-pentaphosphate when incubated with MgATP(-2) and tripolyphosphate or tetrapolyphosphate (P(4)), respectively. Diadenosine 5',5',-P(1),P(4)-tetraphosphate represented the main product when the enzymes were supplied with only MgATP(2-). The At4CL2 mutant M293P/K320L was studied in more detail and was also found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5',5'-P(1),P(5)-pentaphosphate and dAp(4)dA from the appropriate substrates, p(4)A and dATP, respectively. Formation of Ap(3)A from ATP and ADP was not observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in both p(4)A and diadenosine 5',5',-P(1),P(4)-tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.     

Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases.

Diadenosine polyphosphates (ApnAs) act as extracellular signaling molecules in a broad variety of tissues. They were shown to be hydrolyzed by surface-located enzymes in an asymmetric manner, generating AMP and Apn-1 from ApnA. The molecular identity of the enzymes responsible remains unclear. We analyzed the potential of NPP1, NPP2, and NPP3, the three members of the ecto-nucleotide pyrophosphatase/phosphodiesterase family, to hydrolyze the diadenosine polyphosphates diadenosine 5',5"'-P1,P3-triphosphate (Ap3A), diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), and diadenosine 5',5"'-P1,P5-pentaphosphate, (Ap5A), and the diguanosine polyphosphate, diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). Each of the three enzymes hydrolyzed Ap3A, Ap4A, and Ap5A at comparable rates. Gp4G was hydrolyzed by NPP1 and NPP2 at rates similar to Ap4A, but only at half this rate by NPP3. Hydrolysis was asymmetric, involving the alpha,beta-pyrophosphate bond. ApnA hydrolysis had a very alkaline pH optimum and was inhibited by EDTA. Michaelis constant (Km) values for Ap3A were 5.1 micro m, 8.0 micro m, and 49.5 micro m for NPP1, NPP2, and NPP3, respectively. Our results suggest that NPP1, NPP2, and NPP3 are major enzyme candidates for the hydrolysis of extracellular diadenosine polyphosphates in vertebrate tissues.     

Magic-angle spinning (31)P NMR spectroscopy of condensed phosphates in parasitic protozoa: visualizing the invisible

We report the results of a solid-state (31)P nuclear magnetic resonance (NMR) spectroscopic investigation of the acidocalcisome organelles from Trypanosoma brucei (bloodstream form), Trypanosoma cruzi and Leishmania major (insect forms). The spectra are characterized by a broad envelope of spinning sidebands having isotropic chemical shifts at approximately 0, -7 and -21 ppm. These resonances are assigned to orthophosphate, terminal (alpha) phosphates of polyphosphates and bridging (beta) phosphates of polyphosphates, respectively. The average polyphosphate chain length is approximately 3.3 phosphates. Similar results were obtained with whole L. major promastigotes. (31)P NMR spectra of living L. major promastigotes recorded under conventional solution NMR conditions had spectral intensities reduced with respect to solution-state NMR spectra of acid extracts, consistent with the invisibility of the solid-state phosphates. These results show that all three parasites contain large stores of condensed phosphates which can be visualized by using magic-angle spinning NMR techniques.     

Hydrolysis of synthetic pyrophosphoric esters by an isoenzyme of apyrase from Solanum tuberosum.

A highly purified isoenzyme of apyrase obtained from potatoes (Solanum tuberosum var. Pimpernel) exhibits a low specificity for the organic moiety of synthetic pyro- and triphosphates. Methyl di- and tri-phosphates were hydrolysed at higher rates than ADP and ATP, but their Km values were also higher. Steric hindrance at the carbon atom linked to the pyrophosphate chain decreases both binding and maximum rate, whereas length or polarity of the organic chain do not have systematic effects. t-Butyl diphosphate, inorganic pyrophosphate, adenosine 5'-[alpha,beta-methylene]triphosphate and adenosine 5'-[beta,gamma-methylene]triphosphate are competitive inhibitors of the hydrolysis of ATP and ADP.     

CHROMATOGRAPHIC ANALYSES OF ACID-SOLUBLE POLYPHOSPHATES IN CHLORELLA CELLS

Using uniformly ³²P-labeled Chlorella cells as material, compositionof acid-soluble inorganic polyphosphates was studied by paperchromatography and ion-exchange chromatography. 2.By the paper chromatographic analysis it was found that theacid-soluble polyphosphates consisted of highly condensed polyphosphates.Ring-forming tri- and tetrametaphosphates, pyrophosphate andtripolyphosphate were not detected in the acid-soluble fractionof the algal cells. 3.By an ion-exchange chromatography with the use of increasingconcentrations of KCl-solution as eluant, it was found thatthe acid-soluble polyphosphate was a mixture of polyphosphateswith a variety of condensation number (n-values). Polyphosphatesof the n-values between 3 and 15 were only 20% of the totalacid-soluble polyphosphate. The majority of the other polyphospateshad greater n-values which was eluted with 0.5–1.0 M KCl. (Received March 2, 1964; )     

The Saccharomyces cerevisiae YOR163w gene encodes a diadenosine 5', 5"'-P1,P6-hexaphosphate (Ap6A) hydrolase member of the MutT motif (Nudix hydrolase) family

The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes of Mr 21,443. By cloning and expressing this gene in Escherichia coli and S. cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity. Diadenosine 5',5"'-P1, P6-hexaphosphate is the preferred substrate, and hydrolysis in H218O shows that ADP and adenosine 5'-tetraphosphate are produced by attack at Pbeta and AMP and adenosine 5'-pentaphosphate are produced by attack at Palpha with a Km of 56 microM and kcat of 0.4 s-1. Diadenosine 5',5"'-P1,P5-pentaphosphate, adenosine 5'-pentaphosphate, and adenosine 5'-tetraphosphate are also substrates, but not diadenosine 5',5"'-P1,P4-tetraphosphate or other dinucleotides, mononucleotides, nucleotide sugars, or nucleotide alcohols. The enzyme, which was shown to be expressed in log phase yeast cells by immunoblotting, displays optimal activity at pH 6.9, 50 degrees C, and 4-10 mM Mg2+ (or 200 microM Mn2+). It has an absolute requirement for a reducing agent, such as dithiothreitol (1 mM), and is inhibited by Ca2+ with an IC50 of 3.3 mM and F- (noncompetitively) with a Ki of 80 microM. Its function may be to eliminate potentially toxic dinucleoside polyphosphates during sporulation.     

31P NMR spectroscopy of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. Evidence for high levels of condensed inorganic phosphates

High resolution (31)P nuclear magnetic resonance spectra at 303.6 MHz (corresponding to a (1)H resonance frequency of 750 MHz) have been obtained of perchloric acid extracts of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, the causative agents of African sleeping sickness, Chagas' disease, and leishmaniasis. Essentially complete assignments have been made based on chemical shifts and by direct addition of authentic reference compounds. The results indicate the presence of high levels of short chain condensed polyphosphates: di-, tri-, tetra-, and pentapolyphosphate. (31)P NMR spectra of purified T. brucei, T. cruzi, and L. major acidocalcisomes, calcium and phosphorus storage organelles, indicate that polyphosphates are abundant in these organelles and have an average chain length of 3.11-3.39 phosphates. In the context of the recent discovery of several pyrophosphate-utilizing enzymes in trypanosomatids, the presence of these inorganic polyphosphates implies a critical role for these molecules in these parasites and a potential new route to chemotherapy.     

Steric and charge factors in the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase hydrolysis.

The contribution of steric and negative charge factors to the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase was investigated. The hydrolysis rates of uridylyl(3'-5')N6-(N-threonylcarbonyl)-adenosine, its model derivatives, methyl ester and O-benzyl ester, together with unmodified uridyly (3'-5')adenosine, were studied. It was found that the contribution of both factors is of the same order. The steric inhibition of digestion is distinctly higher than that confirmed by N6-(delta2-isopentenyl)adenosine [1], which is ascribed to the rigid conformation of the threonylcarbonyladenosine side chain.