Short-chain Polyphosphates Research Articles

ZDDP Tribofilm Formation and Removal

While ZDDP tribofilm formation has been widely studied, the mechanism of ZDDP tribofilm removal during rubbing is still unclear. The study employs a ball on disc tribometer to monitor ZDDP tribofilm development in rolling-sliding, mixed lubrication conditions. It is found that when ZDDP tribofilms are formed very rapidly, as is the case with short alkyl chain, secondary ZDDPs, a large proportion of the initially-formed tribofilm is suddenly lost during rubbing. By contrast, the tribofilms that form more slowly from primary ZDDPs and longer chain secondaries are not partially lost during rubbing. XPS analysis showed that a rapidly-formed tribofilm before its partial removal has a very small Zn/O ratio, and a high BO/NBO. This suggests that such tribofilm contains a significant proportion of ultraphosphate, which is likely to have a relatively weak structure due to lack of stabilising cations. This may result in the tribofilm being partially removed when it reaches a certain thickness. By comparison, the remaining tribofilm, and also tribofilms that form slowly, have high Zn/O and low BO/NBO. This suggests that they consist of short chain polyphosphates and are thus stronger and more durable.Graphical

Getting Grip on Phosphorus: Potential of Microalgae as a Vehicle for Sustainable Usage of This Macronutrient.

Phosphorus (P) is an important and irreplaceable macronutrient. It is central to energy and information storage and exchange in living cells. P is an element with a "broken geochemical cycle" since it lacks abundant volatile compounds capable of closing the P cycle. P fertilizers are critical for global food security, but the reserves of minable P are scarce and non-evenly distributed between countries of the world. Accordingly, the risks of global crisis due to limited access to P reserves are expected to be graver than those entailed by competition for fossil hydrocarbons. Paradoxically, despite the scarcity and value of P reserves, its usage is extremely inefficient: the current waste rate reaches 80% giving rise to a plethora of unwanted consequences such as eutrophication leading to harmful algal blooms. Microalgal biotechnology is a promising solution to tackle this challenge. The proposed review briefly presents the relevant aspects of microalgal P metabolism such as cell P reserve composition and turnover, and the regulation of P uptake kinetics for maximization of P uptake efficiency with a focus on novel knowledge. The multifaceted role of polyPhosphates, the largest cell depot for P, is discussed with emphasis on the P toxicity mediated by short-chain polyPhosphates. Opportunities and hurdles of P bioremoval via P uptake from waste streams with microalgal cultures, either suspended or immobilized, are discussed. Possible avenues of P-rich microalgal biomass such as biofertilizer production or extraction of valuable polyPhosphates and other bioproducts are considered. The review concludes with a comprehensive assessment of the current potential of microalgal biotechnology for ensuring the sustainable usage of phosphorus.

Short-chain polyphosphates: Extraction effects on migration and size estimation of Saccharomyces cerevisiae extracts with polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis is commonly used to characterize the chain length of polyphosphates (polyP), more generally called condensed phosphates. After separation, nonradioactive, optical polyP staining is limited to chain lengths greater than 15 monomers with toluidine blue or 4',6-diamidino-2-phenylindole. PolyP chain lengths longer than 62 monomers were correlated to the shortest DNA ladders. In this study, synthetic linear polyPs (Sigma-Aldrich "Type 45", estimated mean length of 45 monomers), trimetaphosphate (trimetaP: 3 ring), tripolyphosphate (tripolyP), pyrophosphate (PPi ), and inorganic orthophosphate (o-Pi ) were visualized after separation by an in situ hydrolytic degradation process to o-Pi that was subsequently stained with methyl green. Statistically insignificant migration reduction of synthetic short-chain polyP after perchloric acid or phenol-chloroform extraction was confirmed with the Friedman test. 31 P diffusion-ordered NMR spectroscopy confirmed that extraction also reduced PPi diffusivity by <10%. Linear regression between the Rf peak migration value and the logarithm of synthetic polyP molecular weights enabled estimation of extracted polyP chain lengths from 2 to 45 monomers. Linear polyP extracts from Saccharomyces cerevisiae grown in aerobic conditions were generally shorter than extracts cultured in anaerobic conditions. Extractions from both aerobic and anaerobic S. cerevisiae included tripolyP and o-Pi , but no PPi .

Bacterial polyphosphates induce CXCL4 and synergize with complement anaphylatoxin C5a in lung injury.

Polyphosphates are linear polymers of inorganic phosphates that exist in all living cells and serve pleiotropic functions. Bacteria produce long-chain polyphosphates, which can interfere with host defense to infection. In contrast, short-chain polyphosphates are released from platelet dense granules and bind to the chemokine CXCL4. Here, we report that long-chain polyphosphates induced the release of CXCL4 from mouse bone marrow-derived macrophages and peritoneal macrophages in a dose-/time-dependent fashion resulting from an induction of CXCL4 mRNA. This polyphosphate effect was lost after pre-incubation with recombinant exopolyphosphatase (PPX) Fc fusion protein, demonstrating the potency of long chains over monophosphates and ambient cations. In detail, polyphosphate chains >70 inorganic phosphate residues were required to reliably induce CXCL4. Polyphosphates acted independently of the purinergic P2Y1 receptor and the MyD88/TRIF adaptors of Toll-like receptors. On the other hand, polyphosphates augmented LPS/MyD88-induced CXCL4 release, which was explained by intracellular signaling convergence on PI3K/Akt. Polyphosphates induced Akt phosphorylation at threonine-308. Pharmacologic blockade of PI3K (wortmannin, LY294002) antagonized polyphosphate-induced CXCL4 release from macrophages. Intratracheal polyphosphate administration to C57BL/6J mice caused histologic signs of lung injury, disruption of the endothelial-epithelial barrier, influx of Ly6G+ polymorphonuclear neutrophils, depletion of CD11c+SiglecF+ alveolar macrophages, and release of CXCL4. Long-chain polyphosphates synergized with the complement anaphylatoxin, C5a, which was partly explained by upregulation of C5aR1 on myeloid cells. C5aR1-/- mice were protected from polyphosphate-induced lung injury. C5a generation occurred in the lungs and bronchoalveolar lavage fluid (BALF) of polyphosphate-treated C57BL/6J mice. In conclusion, we demonstrate that polyphosphates govern immunomodulation in macrophages and promote acute lung injury.

Immune escape by polyphosphate?

Host Defense Polyphosphates are inorganic linear polymers with diverse biological functions. In mammals, low–molecular weight short-chain polyphosphates are sequestered within platelets and mast cell secretory dense granules and act as signaling molecules on release. Bacteria such as Escherichia coli can also synthesize high–molecular weight long-chain polyphosphates that can serve as, among other things, energy and phosphate reserves. Roewe et al. report that polyphosphates in E. coli impaired survival in a mouse model of sepsis. In vitro, exposure to bacterial polyphosphates hinders lipopolysaccharide (LPS)–induced polarization of macrophages into a proinflammatory M1 phenotype. Instead, an anti-inflammatory M2 signature is favored, in part caused by suppression of LPS-TLR4–induced interferon β production. Although incompletely understood, E. coli polyphosphates antagonize antimicrobial macrophage functions such as phagocytosis, nitric oxide synthesis, and inflammatory cytokine production. Nat. Commun. 11 , 4035 (2020).

Inorganic Polyphosphate and Physiological Properties of Saccharomyces cerevisiae Yeast Overexpressing Ppn2.

The effect of the yeast endopolyphosphatase Ppn2 overproduction on the metabolism of inorganic polyphosphates in Saccharomyces cerevisiae yeast was studied. Expression of the PPN2 gene under control of the strong constitutive promoter of glyceraldehyde 3-phosphate dehydrogenase gene (PKG1) led to a significant increase in the endopolyphosphatase activity stimulated by cobalt/zinc ions. This activity was present in both soluble and membrane subcellular fractions; it was higher toward long-chain polyphosphates and could be stimulated by ADP. The content of short-chain polyphosphates in the cells of the overexpressing strain was ~2.5 times higher compared to the parent strain. The cells overexpressing Ppn2 were more resistant to peroxide and alkali. The role of short-chain polyphosphates in the adaptation to these stress factors is discussed.

Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI.

IntroductionFactor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin.Methods and ResultsPurified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma.ConclusionsOur data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP.

Speciation Dynamics of Phosphorus during (Hydro)Thermal Treatments of Sewage Sludge.

(Hydro)thermal treatments of sewage sludge from wastewater treatment process can significantly reduce waste volume and transform sludge into valuable products such as pyrochar and hydrochar. Given the global concern with phosphorus (P) resource depletion, P recycling/reclamation from or direct soil application of the derived chars can be potential P recycling practices. In order to evaluate P recyclability as well as the selection and optimization of treatment techniques, it is critical to understand the effects of different treatment techniques and conditions on P speciation and distribution. In the present study, we systematically characterized P speciation in chars derived from thermal (i.e., pyrolysis) and hydrothermal treatments of municipal sewage sludge using complementary chemical extraction and nuclear magnetic resonance (NMR) spectroscopy methods. P species in the raw activated sludge was dominated by orthophosphate and long-chain polyphosphates, whereas increased amounts of pyrophosphate and short-chain polyphosphates formed after pyrolysis at 250-600 °C. In contrast, hydrothermal treatments resulted in the production of only inorganic orthophosphate in the hydrochar. In addition to the change of molecular speciation, thermal treatments also altered the physical state and extractability of different P species in the pyrochars from pyrolysis, with both total P and polyphosphate being less extractable with increasing pyrolysis temperature. Results from this study suggest that P speciation and availability in sludge-derived chars are tunable by varying treatment techniques and conditions, and provide fundamental knowledge basis for the design and selection of waste management strategies for better nutrient (re)cycling and reclamation.

High functional diversity within species of arbuscular mycorrhizal fungi is associated with differences in phosphate and nitrogen uptake and fungal phosphate metabolism.

Plant growth responses following colonization with different isolates of a single species of an arbuscular mycorrhizal (AM) fungus can range from highly beneficial to detrimental, but the reasons for this high within-species diversity are currently unknown. To examine whether differences in growth and nutritional benefits are related to the phosphate (P) metabolism of the fungal symbiont, the effect of 31 different isolates from 10 AM fungal morphospecies on the P and nitrogen (N) nutrition of Medicago sativa and the P allocation among different P pools was examined. Based on differences in the mycorrhizal growth response, high, medium, and low performance isolates were distinguished. Plant growth benefit was positively correlated to the mycorrhizal effect on P and N nutrition. High performance isolates increased plant biomass by more than 170 % and contributed substantially to both P and N nutrition, whereas the effect of medium performance isolates particularly on the N nutrition of the host was significantly lower. Roots colonized by high performance isolates were characterized by relatively low tissue concentrations of inorganic P and short-chain polyphosphates and a high ratio between long- to short-chain polyphosphates. The high performance isolates belonged to different morphospecies and genera, indicating that the ability to contribute to P and N nutrition is widespread within the Glomeromycota and that differences in symbiotic performance and P metabolism are not specific for individual fungal morphospecies.

Antibakteriální účinky komerčních fosfátů na vybrané mikroorganismy

In the food industry, phosphates, polyphosphates and their salts are used, for example, as emul­si­fying agents in the production of processed cheese. The inhibitory effects of three commercially avai­la­ble phosphates and polyphosphates differing in their chain length (690, S9 and HBS) were tested on a set of 15 gram-positive or gram-negative CCM (Czech Collection of Microorganisms) strains and on 12 bacterial strains isolated from processed cheeses. Five different concentrations of each phosphate were chosen (0.1, 0.2, 0.3, 0.4 and 0.5% w/v) in order to observe the inhibitory effects of the phosphate salts on the growth of the microorganisms tested. Sensitivity of the individual bacterial strains to phosphates was observed of a liquid cultivation medium which was supplemented with applied salts. Subsequently, the growth in cells was determined by measuring optical density at a wavelength of 600 nm. According to the results, 690 and S9 phosphates, containing mainly orthophosphates, diphosphates (pyrophosphates) and short-chain polyphosphates, do not have a significant inhibitory effect on the growth of the tested bacteria. Significant inhibitory effects were observed only in HBS salt (a mixture of long-chain polyphosphates), which showed antibacterial effects on all gram-positive bacteria tested (both the CCM strains and those isolated from processed cheeses). The antibacterial effect of phosphates on gram-positive microorganisms is growing with the increasing length of the polyphosphate chain. This study has not proved a significant effect of the phosphates tested on the growth of gram-negative bacteria used.

An analytical study of tribofilms generated by the interaction of ashless antiwear additives with ZDDP using XANES and nano-indentation

Mixtures of zinc dialkyl dithiophosphates (ZDDP) and ashless fluorothiophosphates and thiophosphates were examined under tribological conditions. The tribofilms were studied using nano-indentation, electron microscopy and X-ray absorption near edge structure spectroscopy. With the addition of ashless fluorothiophosphates and thiophosphates, there is significant reduction in wear at identical phosphorous levels. Tribofilms formed with mixtures of ZDDP and ashless antiwear additives had stable pad-like structures, while those from ZDDP alone were more heterogeneous. When ZDDP alone is used, S L-edge indicates a preponderance of sulfates at the surface of the tribofilms, while in the presence of a mixture of antiwear additives a mixture of sulfates and sulfides was observed. In all the cases, the phosphates formed were short chain polyphosphates of Zn.

H-Prune through GSK-3β interaction sustains canonical WNT/β-catenin signaling enhancing cancer progression in NSCLC.

H-Prune hydrolyzes short-chain polyphosphates (PPase activity) together with an hitherto cAMP-phosphodiesterase (PDE), the latest influencing different human cancers by its overexpression. H-Prune promotes cell migration in cooperation with glycogen synthase kinase-3 (Gsk-3β). Gsk-3β is a negative regulator of canonical WNT/β-catenin signaling. Here, we investigate the role of Gsk-3β/h-Prune complex in the regulation of WNT/β-catenin signaling, demonstrating the h-Prune capability to activate WNT signaling also in a paracrine manner, through Wnt3a secretion. In vivo study demonstrates that h-Prune silencing inhibits lung metastasis formation, increasing mouse survival. We assessed h-Prune levels in peripheral blood of lung cancer patients using ELISA assay, showing that h-Prune is an early diagnostic marker for lung cancer. Our study dissects out the mechanism of action of h-Prune in tumorigenic cells and also sheds light on the identification of a new therapeutic target in non-small-cell lung cancer.

Yolk hydrolases in the eggs of Anticarsia gemmatalis hubner (Lepidoptera: Noctuidae): A role for inorganic polyphosphate towards yolk mobilization

Despite being the main insect pest on soybean crops in the Americas, very few studies have approached the general biology of the lepidopteran Anticarsia gemmatalis and there is a paucity of studies with embryo formation and yolk mobilization in this species. In the present work, we identified an acid phosphatase activity in the eggs of A. gemmatalis (agAP) that we further characterized by means of biochemistry and cell biology experiments. By testing several candidate substrates, this enzyme proved chiefly active with phosphotyrosine; in vitro assays suggested a link between agAP activity and dephosphorylation of egg yolk phosphotyrosine. We also detected strong activity with endogenous and exogenous short chain polyphosphates (PolyP), which are polymers of phosphate residues involved in a number of physiological processes. Both agAP activity and PolyP were shown to initially concentrate in small vesicles clearly distinct from typically larger yolk granules, suggesting subcellular compartmentalization. As PolyP has been implicated in inhibition of yolk proteases, we performed in vitro enzymatic assays with a cysteine protease to test whether it would be inhibited by PolyP. This cysteine protease is prominent in Anticarsia egg homogenates. Accordingly, short chain PolyP was a potent inhibitor of cysteine protease. We thereby suggest that PolyP hydrolysis by agAP is a triggering mechanism of yolk mobilization in A. gemmatalis.

An XPS Study on the Composition of Zinc Dialkyl Dithiophosphate Tribofilms and Their Effect on Camshaft Lobe Wear

In this study, various zinc dialkyl dithiophosphate (ZDDP)-derived tribofilms were analyzed with X-ray photoelectron spectroscopy (XPS) to determine the relationship between tribofilm chemical composition and wear severity. Samples were generated on camshaft lobes from engine tests conducted at different oil temperatures, and wear depths were measured with a surface profilometer. Tribofilm specimens were analyzed by XPS to assess changes in film chemistry as a function of wear severity. Tribofilms present on all camshaft lobes were found to contain polyphosphate glass of various chain lengths. Long-chain polyphosphates were observed on low wear tracks, whereas short-chain polyphosphates were found on more severe wear tracks. Variations in polyphosphate chain length are also apparent at different wear depths along a low wear track. The presence of long-chain polyphosphates on low wear tracks was indicative of their superior antiwear properties, whereas the occurrence of short-chain polyphosphates on more pronounced wear tracks was indicative of their inferior antiwear properties.

Short Polyphosphates Inhibit the Procoagulant Activities of Long-Chain Polyphosphate: A Novel Role for Platelet-Derived Pyrophosphate?.

AbstractAbstract 1140 Introduction:Inorganic polyphosphates are negatively charged, linear phosphate polymers that influence hemostasis via accelerating factor V activation, triggering the contact pathway, and enhancing fibrin polymerization. The latter two effects require long-chain polyphosphates for optimal activity (>1000mers for activating the contact pathway and >250mers for enhancing fibrin polymerization). We explored whether short polyphosphates could inhibit the procoagulant effects of long polyphosphate polymers. Methods:Polyphosphate was size-fractionated by gel electrophoresis. “Long-chain polyphosphate” (LCP) was a heterogeneous polyphosphate preparation containing polymers ranging from 400 to several thousand phosphates long. Short-chain polyphosphate preparations were narrow fractions of homogeneous size. We also tested additional phosphate-containing molecules including ADP, ATP, monophosphate, pyrophosphate (PPi), and triphosphate. Clotting assays were performed using pooled normal plasma spiked with 0–20 μM LCP and 0–500 μM small (poly)phosphate; clotting was initiated by factor Xa plus CaCl2, or by CaCl2 alone (contact pathway). Fibrin was formed by clotting 2.6 mg/ml fibrinogen with 10 nM thrombin in the presence of 0–500 μM small (poly)phosphate plus 150 μM LCP and CaCl2. Results:Small polyphosphates (size range 23–83mer) reduced the ability of LCP to trigger the contact pathway of blood clotting, while monophosphate, PPi, triphosphate, ADP, and ATP all failed to influence the procoagulant activity of LCP. None of the small (poly)phosphates antagonized the ability of LCP to enhance factor V activation. Interestingly, monophosphate, PPi and triphosphate all inhibited the ability of LCP to enhance fibrin clot structure, with PPi being the most potent. Although ADP and ATP contain di- and tri-phosphates, they did not recapitulate the potent inhibitory activity of PPi. On the other hand, PPi had no measurable effect on the turbidity of fibrin clots formed in the absence of LCP. Conclusions:Short-chain polyphosphates inhibit the ability of LCP to initiate the contact pathway of coagulation. Platelet dense granules contain abundant PPi which is secreted in response to platelet agonists, although biological roles for platelet-secreted PPi are unclear. We propose that PPi is a novel modulator of fibrin clot structure, acting to regulate the effects of longer-chain polyphosphates on fibrin fibril formation. Disclosures:Smith:University of Illinois: Patents & Royalties. Morrissey:University of Illinois: Patents & Royalties.

Chemical Reactivity of Triphenyl Phosphorothionate (TPPT) with Iron: An ATR/FT-IR and XPS Investigation

The surface reactivity of triphenyl phosphorothionate (TPPT) on air-oxidized iron surfaces has been studied by in situ attenuated total reflection Fourier transform infrared spectroscopy (ATR/FT-IR) and ex situ X-ray photoelectron spectroscopy (XPS). The ATR/FT-IR spectra indicate that a reaction took place at 423 K on the iron-coated germanium ATR crystal with the formation of pyrophosphates, organophosphates, and sulfates. The oxidation of the base oil (poly-α-olefin, PAO) during thermal testing, leading to the production of oxygenated compounds that can adsorb onto the iron surface and react with it, resulted in the presence of carbonates and carboxylates in the film. As the heating time increased, the corrosion of the iron coating by the aggressive species formed during the thermooxidative aging of PAO provided the cations necessary for the formation of orthophosphate compounds. The subsequent ex situ XPS analysis, besides substantiating the formation of short-chain polyphosphates, organophosphates, a...

Exopolyphosphatases PPX1 and PPX2 from Corynebacterium glutamicum

Corynebacterium glutamicum accumulates up to 300 mM of inorganic polyphosphate (PolyP) in the cytosol or in granules. The gene products of cg0488 (ppx1) and cg1115 (ppx2) were shown to be active as exopolyphosphatases (PPX), as overexpression of either gene resulted in higher exopolyphosphatase activities in crude extracts and deletion of either gene with lower activities than those of the wild-type strain. PPX1 and PPX2 from C. glutamicum share only 25% identical amino acids and belong to different protein groups, which are distinct from enterobacterial, archaeal, and yeast exopolyphosphatases. In comparison to that in the wild type, more intracellular PolyP accumulated in the Deltappx1 and Deltappx2 deletion mutations but less when either ppx1 or ppx2 was overexpressed. When C. glutamicum was shifted from phosphate-rich to phosphate-limiting conditions, a growth advantage of the deletion mutants and a growth disadvantage of the overexpression strains compared to the wild type were observed. Growth experiments, exopolyphosphatase activities, and intracellular PolyP concentrations revealed PPX2 as being a major exopolyphosphatase from C. glutamicum. PPX2(His) was purified to homogeneity and shown to be active as a monomer. The enzyme required Mg2+ or Mn2+ cations but was inhibited by millimolar concentrations of Mg2+, Mn2+, and Ca2+. PPX2 from C. glutamicum was active with short-chain polyphosphates, even accepting pyrophosphate, and was inhibited by nucleoside triphosphates.

Characterization of Tribofilms from Zinc Dialkyldithiophosphate in the Presence of Overbased Calcium Phenate Detergents

The effect of overbased calcium phenate detergents on the chemical composition of tribofilms from zinc dialkyldithiophosphate (ZDDP) under boundary lubrication has been studied using P, S, Ca, and C X-ray absorption near edge structure (XANES) spectroscopy. Boundary lubrication was achieved by a pin-on-disk reciprocating wear machine. Calcium phenate detergents, in both high and low overbased forms, were used in combination with ZDDP. C K-edge XANES showed that when higher overbased detergents were used the micelles containing calcium carbonate broke down and calcium carbonate was deposited in the tribofilm. Direct evidence for the formation of calcium phosphate from ZDDP in combination with detergents was provided by XANES spectroscopy at the P-, K- and L- edges. Zn2+ ions were replaced by Ca2+ in the polyphosphate structure of tribofilms forming short chain polyphosphates. The effects of these chemical changes were correlated to the tribological performance of ZDDP/detergents systems.

Human Metastasis Regulator Protein H-Prune is a Short-Chain Exopolyphosphatase

The DHH superfamily human protein h-prune, a binding partner of the metastasis suppressor nm23-H1, is frequently overexpressed in metastatic cancers. From an evolutionary perspective, h-prune is very close to eukaryotic exopolyphosphatases. Here, we show for the first time that h-prune efficiently hydrolyzes short-chain polyphosphates (k cat of 3-40 s (-1)), including inorganic tripoly- and tetrapolyphosphates and nucleoside 5'-tetraphosphates. Long-chain inorganic polyphosphates (>or=25 phosphate residues) are converted more slowly, whereas pyrophosphate and nucleoside triphosphates are not hydrolyzed. The reaction requires a divalent metal cofactor, such as Mg (2+), Co (2+), or Mn (2+), which activates both the enzyme and substrate. Notably, the exopolyphosphatase activity of h-prune is suppressed by nm23-H1, long-chain polyphosphates and pyrophosphate, which may be potential physiological regulators. Nucleoside triphosphates, diadenosine hexaphosphate, cAMP, and dipyridamole (inhibitor of phosphodiesterase) do not affect this activity. Mutation of seven single residues corresponding to those found in the active site of yeast exopolyphosphatase led to a severe decrease in h-prune activity, whereas one variant enzyme exhibited enhanced activity. Our results collectively suggest that prune is the missing exopolyphosphatase in animals and support the hypothesis that the metastatic effects of h-prune are modulated by inorganic polyphosphates, which are increasingly recognized as critical regulators in cells.

Modification of H-ZSM-5 zeolites with phosphorus. 2. Interaction between phosphorus and aluminum studied by solid-state NMR spectroscopy

A suite of one- and two-dimensional solid-state NMR methods was used to follow the complex structural changes in different types of phosphorus species in P-ZSM-5 zeolites that were generated upon treatment of H-ZSM5 zeolites with 0–15% P 2O 5, followed by calcination or calcination and steaming. Through space and through bond 27Al– 31P correlations were used for the first time to study the interaction of phosphorus with the aluminum species. Homogeneous impregnation of phosphorus inside the zeolite channels was observed even at low concentrations. In the calcined samples, the 31P resonances were assigned mainly to orthophosphates, short chain polyphosphates and condensed phosphates. These species were converted into various types of aluminum phosphates with steaming and with the increase in P content. The mechanisms of formation of phosphates and aluminum phosphates are proposed.