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PubMed1991

Effects of transformation with the v-src oncogene on inositol phosphate metabolism in rat-1 fibroblasts. D-myo-inositol 1,4,5,6-tetrakisphosphate is increased in v-src-transformed rat-1 fibroblasts and can be synthesized from D-myo-inositol 1,3,4-trisphosphate in cytosolic extracts.

Rat-1 fibroblasts transformed with the v-src oncogene show a 6-fold increase in the apparent amount of an inositol polyphosphate which has a high performance liquid chromatography (HPLC) elution characteristic of the D/L-myo-inositol 1,4,5,6-tetrakisphosphate enantiomeric pair (Johnson, R.M., Wasilenko, W.J., Mattingly, R.R., Weber, M.J., and Garrison, J.C. (1989) Science 246, 121-124). By chemical and enzymatic analysis, the structure of this compound produced in both normal and v-src-transformed rat-1 fibroblasts has been determined to be principally D-myoinositol 1,4,5,6-tetrakisphosphate (D-Ins(1,4,5,6)P4). Chronic stimulation with endothelin-1 in the presence of Li+ significantly increased the amount of D/L-Ins(1,4,5,6)P4 only in the v-src-transformed rat-1 cells, suggesting that production of this compound may be remotely coupled to long term agonist-induced phosphatidylinositol turnover. Further evidence for such a link is provided by the progressive loss of D-Ins(1,4,5,6)P4 from the normal cells deprived of serum stimulation. To define a possible synthetic pathway for D-Ins(1,4,5,6)P4, cytosolic extracts of normal and v-src-transformed cells were incubated with [3H]inositol polyphosphates, and the reaction products were identified by HPLC elution and chemical analysis. Although inositol 1,3,4-trisphosphate 6-kinase activity was prominent in extracts of both normal and transformed cells, only the cytosol from v-src-transformed cells ultimately formed measurable amounts of D-Ins(1,4,5,6)P4 from [3H]inositol 1,3,4-trisphosphate. Approximately 6% of 0.1 microM inositol 1,3,4-trisphosphate was converted to D-Ins(1,4,5,6)P4 during a 2-h incubation at 37 degrees C. Inositol pentakisphosphate was identified as a likely intermediate in this conversion, and extracts of both normal and transformed cells converted [3H]inositol 1,3,4,5,6-pentakisphosphate to D-Ins(1,4,5,6)P4. The synthetic pathway described is consistent with the long term regulation of D/L-Ins(1,4,5,6)P4 levels in rat-1 fibroblasts seen in response to src transformation, serum withdrawal, and chronic endothelin treatment, and identifies several new potential interactions between the pathways of inositol polyphosphate metabolism and those of src transformation.

Animals↗
PubMedDate not supplied

[Effect of surface-active agents (tween-21) on indices of energy metabolism in oleandomycin producers].

The specific growth rate of Streptomyces antibioticus, a producer of oleandomycin, and the specific rate of the antibiotic accumulation in the culture medium during fermentation were investigated. On the basis of the results obtained the fermentation period was divided into 7 phases of development. The culture treated with the surfactant (Tween-21) is characterized by a higher specific growth rate during the whole fermentation and a higher specific rate of the antibiotic accumulation at the stage of the highest production as compared to the control. The ATP content, the value of the adenylate energy charge and the contents of high-molecular weight polyphosphates in the mycelium were examined. In the phase of the intensive growth St. antibioticus was characterized by a higher ATP level and a higher energy charge. More active accumulation of polyphosphates was observed in the late intensive growth phase. It was also found that after the treatment of the culture with Tween-21 it utilized polyphosphates more actively during the antibiotic biosynthesis.

Culture Media↗
PubMedDate not supplied

[Carbohydrate metabolic characteristics of coryneform bacteria].

The peculiarities of glucose metabolism were studied in typical representatives of coryneform bacteria, and its relation to inorganic polyphosphate metabolism was shown. The activity of the first two enzymes in the pentose pathway was found to be low. The key enzymes of glycolysis were detected, viz. fructose-1,6-diphosphate aldolase and 3-phosphoglyceraldehyde dehydrogenase. The second enzyme was more active in Nocardia sp. B-293 similar in its properties to Nocardia erythropolis as compared to the enzymes of the pentose shunt. The existence of polyphosphate glucokinase more active than ATP-dependent hexokinase is indicative of inorganic polyphosphates being involved in the metabolism of the above microorganisms.

Actinomycetales↗
PubMedDate not supplied

[Study of nucleotide biosynthesis by cultured Brevibacterium ammoniagenes using P31 nuclear magnetic resonance spectroscopy].

Using P31 nuclear magnetic resonance, transformations of phosphorous compounds by the culture Brevibacterium ammoniagenes ATCC 6872 in the course of fermentation aimed at the synthesis of nicotinamide adenine dinucleotide were investigated. It was found that the NAD synthesis by the cells treated with surface active substances was markedly influenced by the presence of a small (initial) ATP quantity and was unaffected by an addition of the synthetic polyphosphate (n-40). It was demonstrated that the cells which were not treated with surface active substances contained orthophosphate, carbohydrate monophosphates, and minor quantities of nucleoside triphosphates, polyphosphates, mono- and dinucleotides. After treatment with surface active substances the cells lost these compounds. It is suggested that polyphosphates of the cell and adenosine monophosphate synthesized during its autolysis may enhance the recovery of the ATP level required to trigger glycolysis which serves as the major producer of ATP in the absence of oxidative phosphorylation.

Adenosine Triphosphate↗
PubMed1993

Modulation by protein kinase C of arachidonic acid release from permeabilised myometrial cells of guinea pig uterus.

The effects of protein kinase C activation on phospholipase A2 and phospholipase C activity in permeabilised cultured myometrial cells from guinea pig uterus have been studied. Phospholipase A2 activity was followed by measurement of [3H]arachidonic acid release from [3H]arachidonic acid-prelabelled membrane lipids. [3H]Arachidonic acid release was stimulated by Ca2+ at 1-10 microM and by GTP gamma S at 1 microM to 1 mM in the presence of 10 microM Ca2+. The activation by calcium was enhanced 89.5 +/- 12.7% (P < 0.01) in the presence of 1 microM phorbol 12-myristate 13-acetate (PMA) and that by 1 microM GTP gamma S by 65.4 +/- 4.4% (P < 0.001). The PMA enhancement of arachidonic acid release was completely blocked by 3 microM staurosporine. Phospholipase C activation was followed by measurement of [3H]inositol polyphosphate production from [3H]inositol-prelabelled membrane lipids. This was stimulated by Ca2+ at 0.1 and 10 microM and by 1 and 50 microM GTP gamma S. PMA at 1 microM caused a consistent reduction in the extent of Ca2+ and GTP gamma S-stimulated inositol polyphosphate production and 3 microM reversed the inhibitory action of PMA. The data are consistent with arachidonic acid release in permeabilised myometrial cells from guinea pigs reflecting in large part phospholipase A2 activation and with that pathway being stimulated by protein kinase C activation. They are also consistent with protein kinase C activation causing reduction in phospholipase C pathways in uterine myocytes, at least as measured by inositol polyphosphate release.

Alkaloids↗
PubMed1996

Constitutive activation of a phosphoinositidase C-linked G protein in murine fibroblasts decreases agonist-stimulated Ca2+ mobilization.

We compared Ca2+ signaling and inositol polyphosphate metabolism in NIH-3T3 cells stably transfected with cDNA encoding either the wild-type G protein G16 alpha subunit or a GTPase-deficient alpha 16 subunit (Q212L-alpha 16). Constitutive activation of phosphoinositidase C (PIC) in cells expressing Q212L-alpha 16 was demonstrated by 1) an increased basal level of [3H]inositol polyphosphates, 2) an enhanced rate of [3H]inositol polyphosphate accumulation in cells treated with 10 mM LiCl, and 3) an increased rate of incorporation of [3H]inositol into cell lipids. Q212L-alpha 16 cells had a diminished cell growth rate. Basal intracellular Ca2+ concentration was equivalent in Fura-2 acetoxymethyl ester-loaded Q212L-alpha 16 cells compared with controls; however, calcium release in Q212L-alpha 16 cells exposed to ionomycin, ATP (a G protein-linked agonist), or platelet-derived growth factor (a tyrosine kinase-linked agonist) was decreased. Permeabilized, 45Ca-loaded Q212L-alpha 16 cells released less 45Ca at each concentration of inositol-1,4,5-trisphosphate than did control cells. Accordingly, the total amount of inositol trisphosphate (IP3) receptor protein was decreased in Q212L-alpha 16 cells relative to controls. These data demonstrate that Q212L-alpha 16 cells maintain physiological levels of cytoplasmic calcium and partially loaded Ca2+ stores in the face of constitutively active PIC. This is accomplished, at least in part, by down-regulation of IP3 receptor number. Thus, diminution in cell growth rate in Q212L-alpha 16 cells seems to be attributable to a combination of at least two effects: a direct effect of PIC activation leading to partial depletion of Ca2+ stores and an indirect, adaptive response resulting in a decreased IP3 receptor number.

3T3 Cells↗
PubMed2026

Genome-Resolved Metagenomics Revealed the Functional Potential of Core Novel and Known Genera Key to Processes in Full-Scale Aerobic Granular Sludge Plants.

Microbial communities are critical for nutrient removal in aerobic granular sludge (AGS) wastewater treatment plants (WWTPs). Despite the stable long-term operation of full-scale AGS WWTPs, the microbial populations and functional traits sustaining stable long-term performance remain poorly resolved. To address this gap, the recovered MAG catalog from nine full-scale AGS WWTPs across five countries was analyzed. From this catalog, 74 high-quality core MAGs were identified and used for downstream taxonomic characterization and functional analyses. These high-quality core MAGs spanned 48 established and 7 novel genera, representing 31 known and 43 novel species. Functional analysis linked core MAGs to key WWTP processes: polyphosphate accumulation (9), glycogen accumulation (12), denitrification (62), and nitrification (1). These included four novel MAGs with glycogen-accumulating (3) and polyphosphate-accumulating (1) potential and 11 capable of nitrous oxide reduction, critical for mitigating greenhouse gas emissions. Ca. Phosphoribacter was the most abundant genus, highlighting its underestimated role caused by misclassification as Tetrasphaera in 16S rRNA surveys. Specifically, Ca. P. hodrii was the dominant species, exhibiting enhanced sugar uptake and amino acid synthesis as likely drivers of its enrichment in the AGS WWTPs. Overall, this study resolves for the first time the taxa and functional traits consistently enriched in full-scale AGS systems, enabling a shift from an empirical performance assessment toward biologically informed process interpretation.

Sewage↗
PubMed2005

Diversity matters: dynamic simulation of distributed bacterial states in suspended growth biological wastewater treatment systems.

A MATLAB-based program was developed to simulate the distributions of states and behaviors of microbial storage product-accumulating bacteria in suspended growth systems. Currently available computer simulators of these systems predict dynamic behaviors by numerically solving differential biokinetic equations using average, or "lumped" system states (e.g., average microbial storage products concentrations). However, individual bacterial states are expected to diverge from average values, in part because individuals can have different hydrodynamic histories in terms of their residence times in upstream completely mixed flow reactors. The distributed state simulation program presented in this paper (DisSimulator 1.0) tracks individual bacteria as they move through a completely mixed reactor system. The program was evaluated for competition between polyphosphate-accumulating organisms (PAOs) and non-polyphosphate-accumulating heterotrophs in an enhanced biological phosphorus removal (EBPR) system for treatment of domestic wastewater. For identical systems and process conditions, simulations accounting for distributed states predicted larger anaerobic and aerobic solids residence time requirements for successful EBPR than did simulations using the lumped approach. One reason for this was that distributed simulations predicted large numbers of the PAOs were essentially inactive due to depleted or maximized storage product contents, while the lumped simulations predicted homogenous, 100% active PAO populations. Characteristic state profile shapes developed rapidly and were stable as total population numbers changed. Lumped state assumptions were demonstrated to produce large errors in predictions of EBPR system performance, and so consideration of distributed states may improve the accuracy of microbial storage products-based process simulations in systems with completely mixed hydrodynamics.

Bacterial Physiological Phenomena↗
PubMed1979

Continuous production of NADP by immobilized Brevibacterium ammoniagenes cells.

Whole cells of Brevibacterium ammoniagenes IAM 1645 having the polyphosphate NAD-kinase were successfully immobilized in a polyacrylamide gel lattice. The immobilized cells were activated by treatment with organic solvents or detergents. The pH optimum of the immobilized cells for the production of NADP was 7.0, and divalent metal ions were required to maintain the elevated activity of polyphosphate NAD-kinase. Highly pure NADP was continuously produced in high yield by the immobilized cell column. The half-life of this column was about eight days.

Brevibacterium↗
PubMed2005

Role of PRIP-1, a novel Ins(1,4,5)P3 binding protein, in Ins(1,4,5)P3-mediated Ca2+ signaling.

PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-delta1 (PLC-delta1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,5)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Trehalases from spores and vegetative cells of yeast Saccharomyces cerevisiae.

Trehalase (THA) activity from S. cerevisiae spores and vegetative cells could be differentiated in cell-free extracts. THA from the vegetative cells has an optimal activity at neutral pH whereas biphase pH optimum in the spores was observed. The enzyme from the spores exhibited higher thermostability than that from the vegetative cells. The presence of magnesium ions was necessary mainly for THA activity from the vegetative cells. The effect of the other metal ions studied: Hg2+, Ag2+, Cu2+, Fe3+, Ni2+, Cd2+ etc. (Table II), on THA from both sources was almost the same, however, the spores THA was resistant to Pb2+ and especially to Zn2+. Moreover, the influence of inorganic polyphosphates and polyamines was also quite dissimilar. Polyphosphates inhibited THA from the vegetative cells and to a smaller extent from the spores. On the other hand, polyamines stimulated highly THA activity from vegetative yeast cells in contrast to spores one. The effect of these ions modulators would facilitate differentiating of THA activity in the cell-free extracts from both sources. These data could be interpreted as phenotypic reflections of trehalase genes expression in the S. cerevisiae cells.

Culture Media↗
PubMed1981

Accumulation of pyrophosphate and other energy-rich phosphorous compounds under various conditions of yeast growth.

In the cells of hybrid yeast strain Saccharomyces N.C.Y.C. 644 SU3 (Karlsberg collection), a large amount of pyrophosphate (30-300 micro mol per g of dry weight) accumulates whatever the aeration conditions and the contest of glucose in the medium. The content of pyrophosphate is 10-100 times higher than that of ATP. At the early and mid-exponential growth phases two maxima of pyrophosphate accumulation are observable. The periods of maximal pyrophosphate accumulation in yeast coincide with those of the minimal contest of polymeric acid-soluble polyphosphates and intense budding. In the light of the data obtained, the question is discussed as to the relationship between the metabolism of pyrophosphates and acid-soluble polyphosphates in yeats.

Adenosine Triphosphate↗
PubMed1995

Reactions involving carbamyl phosphate in the presence of precipitated calcium phosphate with formation of pyrophosphate: a model for primitive energy-conservation pathways.

The formation of carbamyl phosphate (CAP) in dilute solutions of cyanate (NCO-) and orthophosphate (Pi) was measured both in the absence and in the presence of a precipitated matrix of calcium phosphate (Pi.Ca). The second-order rate constant and the free energy of CAP synthesis were not modified by the presence of the solid matrix, indicating that synthesis occurs in the homogeneous Pi-containing solution. The elimination reaction of CAP to form NCO- and Pi followed first-order kinetics and the rate constant was the same whether or not calcium phosphate was present. Elimination was not complete, and the steady level of remaining CAP was that expected from the free energy of synthesis. The formation of pyrophosphate (PPi) was detected in CAP-containing medium only in the presence of calcium, showing a close correlation with the amount of precipitated Pi.Ca. Phosphorolysis of CAP followed a sigmoidal time course, compatible with adsorption of CAP to the solid matrix as a prelude to transphosphorylation. Addition of 5'-AMP and of short linear polyphosphates inhibited phosphorolysis of CAP. It is proposed that the presence of a solid phosphate matrix and the relative concentrations of cyano compounds, as well as those of nucleotides and inorganic polyphosphates, could have played a crucial role in the conservation of chemical energy of CAP and in its use in prebiotic phosphorylation reactions.

Adenosine Monophosphate↗
PubMed1979

Transport of manganese into Saccharomyces cerevisiae.

The uptake of Mn2+ by Saccharomyces cerevisiae at the expense of endogenous sources of energy depends on the stage of culture development and is maximum in the middle of the exponential phase. The ability of cells to take up Mn+ is related to the content of intracellular potassium at all stages of growth, to the content of ATP during the exponential phase and it is not related to the content of inorganic polyphosphates. The uptake is inhibited by oligomycin (25 microgram/ml) by 50-85% and under anaerobic conditions by 10-50%, depending on the stage of growth, indicating the role of aerobic phosphorylation in the process. The uptake of Mn+ is apparently associated with a hydrolysis of low-molecular weight polyphosphates and ATP, as well as with the exit of K+ from cells.

Adenosine Triphosphate↗
PubMed2006

Convenient synthesis of 3- and 6-deoxy-D-myo-inositol phosphate analogues from (+)-epi- and (-)-vibo-quercitols.

Starting from (+)-epi- and (-)-vibo-quercitols readily produced by bioconversion of myo-inositol, some biologically interesting phosphate and polyphosphate analogues, including the Ins(1,4,5)P(3) derivatives of 3-deoxy- and 6-deoxy-D-myo-inositol, could be readily prepared in a conventional manner. In addition, chemical modification at C-2 of the 3-deoxy Ins(1,4,5)P(3) provided 2-epimer, and 2-deoxy and 2-deoxy-2-fluoro forms. Eight polyphosphate analogues obtained were assayed for biological activity against PDH-Pase and PDH-K, and G6Pase, but none proved positive.

Animals↗
PubMed2005

Formation of volutin granules in Corynebacterium glutamicum.

Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted.

Bacterial Proteins↗
PubMed2005

In situ identification and characterization of the microbial community structure of full-scale enhanced biological phosphorous removal plants in Japan.

Fluorescent in situ hybridization (FISH) and polyphosphate (polyP) staining methods were used to characterize the microbial community structure of 13 activated sludge samples taken from nine different Japanese wastewater treatment plants with and without enhanced biological phosphorous removal (EBPR) activities. FISH with published rRNA-targeted oligonucleotide probes for important bacterial groups involving in the EBPR process revealed that Rhodocyclus-related polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms from a gammaproteobacterial lineage GB were the predominant populations detected, representing 4-18% and 10-31% of EUBmix-stained cells, respectively, in those samples. However, a considerable proportion of Rhodocyclus-related PAO cells were observed with no polyP granules accumulated based on polyP staining. This was further supported by a poor correlation between Rhodocyclus-related PAO population and sludge total phosphorous (TP) contents. In contrast, high correlations between polyP-stained cells and sludge TP contents were observed. In particular, among those polyP-stained cells in samples Ariake_A2O and Nakano_AO, more than 85% of them could not be targeted by probe PAOmix. These non-Rhodocyclus-related PAOs included populations from other bacterial divisions and members of the Betaproteobacteria other than those in Rhodocyclus-related group.

Bacteria↗
PubMed1995

Poly-3-hydroxybutyrate/polyphosphate complexes form voltage-activated Ca2+ channels in the plasma membranes of Escherichia coli.

The lipidic polymer, poly-3-hydroxybutyrate (PHB), is found in the plasma membranes of Escherichia col complexed to calcium polyphosphate (CaPPi). The composition, location, and putative structure of the polymer salt complexes led Reusch and Sadoff (1988) to propose that the complexes function as Ca2+ channels. Here we use bilayer patch-clamp techniques to demonstrate that voltage-activated Ca2+ channels composed of PHB and CaPPi are in the plasma membranes of E. coli. Single channel calcium currents were observed in vesicles of plasma membranes incorporated into planar bilayers of synthetic 1-palmitoyl, 2-oleoyl phosphatidylcholine. The channels were extracted from cells and incorporated into bilayers, where they displayed many of the signal characteristics of protein Ca2+ channels: voltage-activated selective for divalent over monovalent cations, permeant to Ca2+, manner by La3+, Co2+, Cd2+, and Mg2+, in that order. The channel-active extract, purified by size exclusion chromatography, was found to contain only PHB and CaPPi. This composition was confirmed by the observation of comparable single channel currents with complexes reconstituted from synthetic CaPPi and PHB, isolated from E. coli. This is the first report of a biological non-proteinaceous calcium channel. We suggest that poly-3-hydroxybutyrate/calcium polyphosphate complexes are evolutionary antecedents of protein Ca2+ channels.

Calcium Channels↗