Acid-insoluble Polyphosphates Research Articles

Polyphosphates and Polyphosphatase Activity in the Yeast Saccharomyces cerevisiae during Overexpression of the DDP1 Gene.

The effects of overexpression of yeast diphosphoinositol polyphosphate phosphohydrolase (DDP1) having endopolyphosphatase activity on inorganic polyphosphate metabolism in Saccharomyces cerevisiae were studied. The endopolyphosphatase activity in the transformed strain significantly increased compared to the parent strain. This activity was observed with polyphosphates of different chain length, being suppressed by 2mM tripolyphosphate or ATP. The content of acid-soluble and acid-insoluble polyphosphates under DDP1 overexpression decreased by 9 and 28%, respectively. The average chain length of salt-soluble and alkali-soluble fractions did not change in the overexpressing strain, and that of acid-soluble polyphosphate increased under phosphate excess. At the initial stage of polyphosphate recovery after phosphorus starvation, the chain length of the acid-soluble fraction in transformed cells was lower compared to the recipient strain. This observation suggests the complex nature of DDP1 involvement in the regulation of polyphosphate content and chain length in yeasts.

Polyphosphate and siliceous granules in the macroscopic gametophytes of the red alga Porphyra purpurea (Bangiophyceae, Rhodophyta)

Phosphorylated fractions in tissues of starved and fresh plants of Porphyra purpurea, a representative of the Bangiophyceae, were analyzed over a 48 h incubation period in pulse-enriched seawater (15 mM phosphorus and 25 mM nitrogen). Compared to Chondrus crispus, a representative of the structurally and reproductively more complex Florideophyceae, P. purpurea takes up phosphorus much more actively, with a higher turnover rate, in which the orthophosphate fraction is predominant and acid-soluble and acid-insoluble polyphosphates are less significant as storage pools. Presence of cytoplasmic acid-insoluble polyphosphate granules was confirmed by transmission electron microscopy and energy dispersive X-ray microanalysis. The granules in P. purpurea were much smaller (20–110 nm in diameter) than those in C. crispus (around 1 mm, but some larger than 2 mm). Larger granules (290–310 nm) were observed in P. purpurea. They were more uniformly electron-opaque, without the reticulated/globular appearance of typical polyphosphate granules. Energy dispersive X-ray microanalysis demonstrated they were siliceous granules. To our knowledge, this is the first report in algae of such structures whose occurrence and metabolic role remain enigmatic.

Inorganic polyphosphates and phosphohydrolases inHalobacterium salinarium

Halobacterium salinarium grown in a liquid medium consumed up to 75% of phosphates originally present in the growth medium and accumulated up to 100 mumol Pi/g wet biomass by the time it entered the growth retardation phase. The content of acid-soluble oligophosphates in the biomass was maximum at the early stage of active growth and drastically decreased when cells reached the growth-retardation phase. The total content of alkali-soluble and acid-insoluble polyphosphates changed very little throughout the cultivation period (five days). The polyphosphate content of H. salinarium cells was close to that of yeasts and eubacteria. The pyrophosphatase, polyphosphatase, and nonspecific phosphatase activities of H. salinarium cells were several times lower than those of the majority of eubacteria. The specific activity of pyrophosphatase, the most active hydrolase of H. salinarium, gradually increased during cultivation, reaching 540 mU/mg protein by the end of the cultivation period. Half of the total pyrophosphatase activity of this halobacterium was localized in the cytosol. The molecular weight of pyrophosphatase, evaluated by gel filtration, was 86 kDa. The effective Km of this enzyme with respect to pyrophosphate was 115 microM.

Polyphosphate and orthophosphate content during the life cycle ofMyxococcus coralloidesD

Pool changes in the orthophosphate and acid soluble and insoluble polyphosphates of Myxococcus coralloides D were determined during the different stages of its life cycle. The acid-insoluble fraction is the major fraction. Polyphosphate was synthesised during vegetative growth in a liquid medium and during aggregation and fruiting body maturation. During the induction and germination of myxospores both types of polyphosphate were depleted, the acid-insoluble fraction during the myxospore induction and the acid-soluble during germination. The orthophosphate pool remained constant or increased during growth in a liquid medium, increased during growth on solid media and myxospore induction and decreased during myxospore germination.

A simple fractionation method for determination of phosphorus components in phytoplankton: Application to natural populations of phytoplankton in summer surface waters of Tokyo Bay

A simple procedure for fractionation of phosphorus components in natural populations of phytoplankton has been developed. By a combination of a trichloroacetic acid (TCA) extraction, a hot dilute acid treatment, a charcoal treatment and an organic solvent extraction, cellular phosphorus was divided into orthophosphate, nucleotide phosphorus, sugar phosphates, acid-soluble polyphosphates, lipid phosphorus, nucleic acid phosphorus, acid-insoluble polyphosphates, and residual phosphorus. The recovery of phosphorus was 94±5%. This technique was applied to natural populations of phytoplankton in summer surface waters of Tokyo Bay. Nucleic acid phosphorus and orthophosphate were most abundant, and accounted for 30 to 50% of total phytoplankton phosphorus. The nucleic acid phosphorus was proportional to the biomass of phytoplankton, irrespective of ambient nutrient concentrations. The orthophosphate showed the largest change in association with the change in cellular phosphorus content as well as in ambient phosphate. The orthophosphate serves as a phosphorus reserve in the natural populations of phytoplankton in summer surface waters of Tokyo Bay whereSkeletonema costatum was dominant. Acid-insoluble polyphosphates tended to increase when ambient concentration of phosphate was high, but their amounts were almost one order of magnitude lower than that of orthophosphate.

Induction of free radicals in seeds by high intensive flashes and the relevant phosphorus metabolism in the seedling

Influence of high intensive flashes on the yield of free radicals in intact seeds and excised embryonic axis, endosperm, and seed coat, and its resulting effect on seedling growth, total biomass production and phosphorus metabolism in wheat (Triticum aeativum), vetch (Vicia sativa L.) and onion (Allium cepa L.) was studied. Free radicals (f.r.) were formed mainly in seed coat and not in the endosperm. Vetch seeds after irradiation had 20.76 X 1013 f.r. g-1 dry intact seed and 17.30 X 1013 f.r. g-1 dry seed coat. Excised seed coats exposed to irradiation also yielded 17.28 × 1013 f.r. g-1 dry matter. High irradiance “white light” flashes induced more f.r. than a monochromatic one of the same photon content. Red (650 nm), farred (750 nm) and even infra-red (1100 nm) radiation did not initiated f.r. formation but resulted in their decay in samples irradiated earlier by “white”, blue and green parts of the spectrum. Blue irradiation of seeds led to the decrease in the length of shoots and roots in comparison to “white”, green and red irradiation but their biomass increased faster than in the seedlings obtained from non-irradiated or irradiated with “white” and green radiation. The quantity of total acid soluble phosphorus followed a sequence with respect to wavelength of radiation: 436 nm > 650 nm> > 540 nm > non-irradiated > 300–800 nm. Quantity of inorganic phosphorus remained unaffected by different spectral character of radiation. The quantity of organic acid soluble nucleic phosphorus and acid insoluble polyphosphates was higher in samples irradiated with red beams (650 ± 6 nm).

Detection of Polyphosphates and Enzymes of Polyphosphate Metabolism in Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus cells, parasitizing in E. coli, contain a considerable amount of inorganic polyphosphates, 55% of the total pool of which is due to the most polymeric acid-insoluble polyphosphates. B. bacteriovorus contains enzymes participating both in the synthesis and consumption of polyphosphates, i.e. 1,3-diphosphoglycerate: polyphosphate phosphotransferase, polyphosphate glucokinase, polyphosphatase, tripolyphosphatase, pyrophosphatase, acid and alkaline phosphatases. The possible role of high-molecular polyphosphates in the vital activity of the bacterial parasite B. bacteriovorus is discussed.

Über den Einfluß des blauen und roten Spektralbereichs auf Phosphatfraktionen, besonders Polyphosphate, bei Grünalgen

Autotrophic cells of Chlamydomonas reinhardii and Chlorella pyrenoidosa, cultured in blue light, have a significant higher content of phosphate compared with cells cultured in the red light with equal dry mass. The greatest effect was observed in the acid-insoluble polyphosphates. The blue light-dependent enhancement was less pronounced in the acid insoluble stable and the acid soluble labile phosphate. In conditions of nitrogen deficiency the polyphosphates contain nearly all the phosphate additionally incorporated in the blue light. The enhancement of 32P-incorporation, compared with the red light, is delayed and more distinct after about 30 min. Blue light cells have a higher content of ATP. The interaction between the blue light-dependent increase of the ATP- and polyphosphate pool is discussed.

Untersuchungen �ber die Raten der Polyphosphatsynthese durch die Photophosphorylierung bei Ankistrodesmus braunii

Short-time experiments with 32P-labelled phosphate and chase experiments with equally labelled cells were carried out with synchronized algae under conditions of optimum phosphate uptake. In short-time experiments, in the presence as in the absence of CO2, orthophosphate and organic phosphates are rapidly labelled, but their time curves show saturation behaviour after 10 to 20 min. Labelling of polyphosphates proceeds at a constant rate after a short lag period of about 5 min. In equally labelled algae 32P-labelling correspondingly decreases in orthophosphate and in organic phosphates, but increases by about the same amount in the fraction of acid-insoluble polyphosphates. In the presence of external phosphate and in the light, polyphosphates show no visible decay within the 20 min of the chase experiments. A comparison of the two kinds of experiments suggests that polyphosphates are secondary products of photophosphorylation following only after orthophosphate and organic phosphates, probably after ATP. The rates of photophosphorylation are certainly much higher than the rates of labelling in organic phosphates because of the limiting phosphate uptake. Since the polyphosphates show no decay during the time of the experiments their turnover is low and the rates of polyphosphate labelling after a phosphate starvation period, and after the short lag period, can be regarded as approximate rates of polyphosphate synthesis. These rates are lower than the rates of phosphate uptake. In young cells of the synchronous culture phosphate replenishment after a 5-h starvation requires 2 to 3 h. After replenishment or in a culture undisturbed by phosphate starvation, the rates of polyphosphate accumulation, like the rates of phosphate uptake are much lower. In the presence of CO2 they are constant for several hours, if related to culture volume with constant cell number. Polyphosphate accumulation is proportional to phosphate uptake under these conditions amounting to about one third. In the absence of CO2, the rates decrease after 2 to 4 h of CO2-starvation and, like in short-time experiments a large proportion of the phosphate taken up is used for polyphosphate accumulation. The low rates of long-time experiments may represent a steady state between formation and decay of polyphosphates. Since the cells kept in the absence of CO2 are prevented from growing they actually accumulate more polyphosphates per cell volume, per chlorophyll, and per dry weight than the cells in the presence of CO2. The rates of polyphosphate formation are discussed with respect to their turnover in the light observed by other investigators. They are regarded to be a result of competition for ATP together with the orthophosphate pool of the cells, and of the compartmentation. The rates of polyphosphate formation are rather low compared with the probable rates of ATP formation under various conditions of photophosphorylation. Therefore, the formation of polyphosphates is regarded as a process of secondary order of magnitude in the energy metabolism of algal cells.

POLYPHOSPHATE FORMATION IN THE ROOTS OF DESCHAMPSIA FLEXUOSA AND URTICA DIOICA

Since 1961, several authors have reported the extraction of polyphosphates (PolyP)from various higher plant tissues (Miyachi, 1961; Tewari and Singh, 1964; Jeffrey,1964). The last named reported that some 30% of the ^^P absorbed by the roots ofBanksia ornata in 24 hours appeared in the form of acid-insoluble polyphosphates. Hesuggested that their synthesis might be a feature common to plants adapted to long-termsurvival in a phosphate-deficient medium.These reports together with others on the occurrence and physiological significanceof polyphosphates in microorganisms and lower plants (e.g. Harold, 1966), led us toinvestigate the amounts of polyphosphate in the roots of Deschampsia flexuosa, which likeBanksia ornata is a plant of habitats low in phosphate and one that shows little dry weightresponse to added phosphate. Urtica dioica, a species very responsive to phosphate, wasinvestigated for comparison. Some aspects of the phosphate behaviour of these plantshave been reported in a previous paper (Nassery and Harley, 1969).

Synthesis of polyphosphate by rat liver mitochondria

The synthesis and hydrolysis of acid insoluble polyphosphates has been observed in many lower species and the enzymes involved have been partially purified by Kornberg and Hughes. Data also exists indicating that in bacteria under experimental conditions of biosynthetic blockade, e.g., nitrogen deficiency etc., polyphosphates accumulate within cells. Lehninger and others have noted that mammalian mitochondria swell in the presence of mitochondrial substrates and in the absence of ADP but shrink when ATP concentration is maintained at a high level. A reasonable explanation for these observations could be that excess substrate in the absence of adequate concentrations of nucleotide acceptor, activates the turnover of high energy phosphate into a “phosphagen” or polyphosphate pool at a very rapid rate, thus allowing ATP concentration to fall. The data in this report indicate that substrate does markedly stimulate phosphate uptake into a high molecular weight polyphosphate of rat liver mitochondria with a concomitant decrease in ATP concentration.

The effect of temperature shifts on the budding cycle of Saccharomyces cerevisiae

1. 1. Inocula of yeast cultures, Saccharomyces cerevisiae strain 25.8, in the stationary phase were diluted with fresh medium and immediately exposed to a temperature cycle treatment lasting 6 hr. In each cycle the cells were kept at the optimum temperature (33 °C) for 25 min, followed by the temperature shock of 5 min at 49.5 °C. At the commencement of the treatment 90 per cent of the cells were single. After the elapse of 3 hr, however, the cells began to bud (only one bud per cell) and by the end of the treatment the buds had grown to the size of the parent. The result is the formation of paired, enlarged cells. 2. 2. These paired cells appear, cytologically speaking, to be blocked in the same stage of their development. However, upon release from the treatment they do not proceed synchronously to cleavage or budding, as, for example, paired cells produced by X-irradiation (2.85 Kr/hr) [28]. For convenience, we refer to the temperature induced paired cells as pseudosynchronized cells (P-S). The P-S cells show a lag period of three hours at the end of the treatment, followed by a gradually increasing budding and fission activity similar to freshly inoculated normal cells. 3. 3. Biochemical analyses were performed on three stages: the logarithmic phase (growth and division), stationary phase (negligible growth and division), and at the end of the heat treatment (P-S cells, growth without division). 4. 4. An almost 50 per cent reduction in the amounts of RNA, and DNA (per dry weight) was found in the P-S cells when compared to normal log cells. Acid soluble sulfhydryl increased (during the treatment) by about 40 per cent. However, the concentration of sulfhydryl in the stationary phase cells has tripled over the log cell values. The glycogen content was found to be rather constant. 5. 5. Acid soluble labile phosphates and polyphosphates (ppt. at pH 2.5 and 4.5) approximately double during the temperature treatment. Acid insoluble polyphosphates in the stationary phase cells are only about 2 3 of the amount found in the exponentially growing cells and could not be detected at all in the P-S cells. 6. 6. Sedimentation of ribosomal particles isolated from cells of the three stages was studied. One peak (80 S) was found in both stationary and P-S cells, but at a lower concentration in the former. A second ribosomal peak (60 S) was present in the log cells. 7. 7. Biochemical data are discussed in the light of the results reported on paired, enlarged yeast cells produced by irradiation and by chemical blocking agents.

Polyphosphate Metabolism in X-Irradiated Division-Inhibited Yeast Cells

Abstract : To describe more fully an effect of X-radiation upon phosphorus compounds of yeast cells, formation and phosphorus transfer from acid insoluble and soluble polyphosphates were measured by the use of P32. Uptake of P32 into insoluble polyphosphates was rapid and was greater in irradiated than in unirradiated cells. Uptake into soluble polyphosphates occurred at a low rate and was decreased in irradiated cells. When cells were transferred from one growth medium to another before irradiation, the above effects were reversed, and P32 incorporation into nucleic acids was reduced. Incorporation into phospholipids was reduced somewhat in both cases, and incorporation into an acid soluble organic phosphorus fraction was increased. Possible mechanism for these effects are discussed. An increased uptake by starved irradiated cells, as contrasted to starved unirradiated cells, was observed and briefly described. (Author)