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PubMed1975

Polyphosphate levels in nongrowing cells of Saccharomyces mellis as determined by magnesium ion and the phenomenon of "Uberkompensation".

Magnesium ion enhances the maximum amount of polyphosphate that resting phosphate-starved cells of Saccharomyces mellis can store by increasing the length of time the cells will continue assimilating phosphate. The divalent cation has no effect on the rate of formation of polymer. As much as 12 times more polyphosphate is formed in cells incubated in reaction mixtures containing 0.3 M MgCl2 than in the absence of Mg2+. Potassium ion also has an influence on the amount of polyphosphate that phosphate-starved cells can accumulate but the degree of stimulation is not very large. Mg2+ and K+ have no effect on polyphosphate formation or storage in phosphate-satiated cells. Apparently, then, there are two systems for polyphosphate accumulation in S. mellis. Each system is stable in nondividing cells. The one present in phosphate-starved cells seems to be repressible by growth of the organism in media containing orthophosphate. The shift from the derepressed state to the repressed state, or vice versa, occurs only in exponentially dividing cells in appropriate media with 100% of the cells in the new physiological state by the time the cell mass has doubled. It is suggested that the word to describe the phenomenon of the accumulation of higher amounts of polyphosphate in phosphate-starved cells than the steady-state level of phosphate-satiated cells be changed from "uberkompensation" to "magnesium ubertriebung," or "magnesium enhancement."

Cell Division↗

PubMed1992

Existence and alpha 1-adrenergic stimulation of inositol polyphosphates in mammalian heart.

The concentration-response curves and the time course of the effects of phenylephrine (0.01-100 microM) on force of contraction and on inositol polyphosphates in isolated electrically stimulated perfused rat hearts (Langendorff technique) were studied. A nonradiometric high performance liquid chromatography metal dye detection technique was used to determine absolute concentration masses/changes of inositol polyphosphates in heart. Products measured after separation with high performance liquid chromatography were inositol 1,4,5-trisphosphate (1,4,5-IP3), inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4) and its isomer 1,3,4,6-IP4, inositol 1,3,4,5,6-pentakisphosphate (1,3,4,5,6-IP5), and inositol hexakisphosphate (IP6). 1,4,5-IP3 (significant at 10 microM) and both IP4 isomers (significant at 1 microM) increased after alpha-adrenoceptor stimulation, whereas 1,3,4,5,6-IP5 and IP6 remained unaffected. Phenylephrine had a concentration-dependent positive inotropic effect (significant at 1 microM). All effects were antagonized by the alpha 1-adrenoceptor antagonist prazosin (0.1 microM), indicating receptor-mediated effects. In a time course study 1,4,5-IP3 was the first compound to increase significantly, within 1 min after stimulation; this rise was followed by an increase in 1,3,4,5-IP4 beginning within 2 min. The increase in all other inositol polyphosphates was slower (5-10 min). The increase in the force of contraction started at 2 min. For comparison, the effects of the beta-adrenoceptor agonist isoprenaline were studied. Isoprenaline produced a positive inotropic effect similar to that of phenylephrine, but all inositol polyphosphates remained unaffected. In conclusion, for the first time the existence of 1,3,4,5,6-IP5 and IP6 was observed in the heart. However, the physiological role of these inositol polyphosphate isomers in the heart remains to be elucidated, because, from the time course, they appear to have no acute intracellular second messenger function. Increased inositol polyphosphate turnover may be involved in the mechanism(s) whereby alpha 1-adrenoceptor stimulation produces an increase in myocardial force of contraction. Because the increase in 1,4,5-IP3 precedes and that in 1,3,4,5-IP4 coincides with the increase in the force of contraction, 1,4,5-IP3 may initiate and 1,3,4,5-IP4 may maintain the positive inotropic effect of alpha 1-adrenoceptor agonists.

Animals↗

PubMed1988

Properties of inositol polyphosphate 1-phosphatase.

We recently described inositol polyphosphate 1-phosphatase, an enzyme which cleaves the 1-phosphate from inositol 1,4-bisphosphate (Ins(1,4)P2) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) (Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 15946-15952). We have now purified the enzyme to homogeneity from calf brain. The enzyme hydrolyzes 50.3 mumol of Ins(1,4)P2/min/mg protein. The enzyme has an apparent mass of 44,000 daltons as determined both by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that it is monomeric. Lithium ions inhibit Ins(1,3,4)P3 hydrolysis uncompetitively with an apparent Ki of approximately 0.3 mM LiCl. Calcium inhibits hydrolysis of Ins(1,4)P2 and Ins(1,3,4)P3 equally, with approximately 40% inhibition occurring at 1 microM free Ca2+. Rabbit polyclonal antiserum against purified inositol polyphosphate 1-phosphatase was prepared which immunoprecipitates approximately 0.3 milliunits of activity/microliter serum (1 unit = 1 mumol of Ins(1,4)P2 hydrolyzed per min). This antiserum was used to determine the enzyme content in several bovine tissues, all of which had a similar intrinsic specific activity (i.e. approximately 0.3 milliunits/microliter antiserum). Tissues studied included brain, heart, kidney, liver, lung, parotid, spleen, testis, and thymus. Approximately 10-15% of the total inositol polyphosphate 1-phosphatase activity in calf brain homogenates remains in a particulate fraction; antiserum also binds 0.3 milliunits of membrane-associated activity/microliter antiserum. Thus, a single enzyme can account for Ins(1,4)P2 hydrolytic activity in the bovine tissues. Ins(1,3,4)P3 metabolism was also investigated in bovine tissue homogenates. Inositol polyphosphate 1-phosphatase accounts for greater than 80% of the hydrolytic activity in all tissues studied except brain, where inositol polyphosphate 4-phosphatase is the major enzyme that hydrolyzes Ins(1,3,4)P3. The apparent Km of inositol polyphosphate 1-phosphatase for Ins(1,3,4)P3 varies approximately 3-4-fold among the bovine tissues.

Animals↗

PubMed1991

Glucose determination using immobilized polyphosphate glucokinase.

Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate:glucose phosphotransferase) was covalently coupled to collagen-coated silica gel beads. The immobilized enzyme, as a packed-bed reactor, was used to determine glucose in serum and other samples. The method was based on a spectrophotometric measurement of NADPH produced by two consecutive reactions, similar to the hexokinase method. The described approach takes advantage of the greater stability of polyphosphate compared to that of ATP, the greater specificity of polyphosphate glucokinase versus that of hexokinase, and the reusability of the immobilized enzyme. Linearity, precision, and accuracy of the method were tested and found to be very good. The results were linear between 10 and 50 nmol of glucose in a 50-microliter sample and the coefficient of variation was less than 4% in five successive determinations. The recovery of glucose was about 100% after calibration of the method. The results of the measurements correlated well with those obtained with soluble polyphosphate glucokinase (r = 0.997, y = 1.036x - 0.016). The immobilized-enzyme reactor showed good operational stability during a month of use, losing about 12% of its initial activity.

Collagen↗

PubMed1989

Nuclear polyphosphate as a possible source of energy during the sporulation of Physarum polycephalum.

31P NMR spectroscopic analysis of the polyphosphate pool in cellular and nuclear extracts of Physarum polycephalum demonstrates that plasmodia and cysts contain inorganic polyphosphates with an average chain length of about 100 phosphates. However, only during sporulation are these high-molecular-weight polyphosphates degraded to a lower molecular weight corresponding to an average chain length of about 10 phosphates. Since polyphosphates are degraded even in the presence of a sufficiently large pool of inorganic phosphate, produced by intracellular injection, we conclude that the degradation of polyphosphates serves in supplying energy for biosynthesis during sporulation rather than in increasing the availability of phosphate.

Cell Nucleus↗

PubMed1990

Polyphosphate-hydrolysis--a protective mechanism against alkaline stress?

Different microorganisms, including yeast and algae, accumulate large amounts of polyphosphates. However, the physiological role of polyphosphates is largely unknown. In vivo 31P NMR studies, carried out in the unicellular alga, Dunaliella salina, demonstrate the cytoplasmic alkalization induces massive hydrolysis of polyphosphates, which is correlated kinetically with the recovery of cytoplasmic pH. Analysis of acid extracts of the cells indicates that long-chain polyphosphates are hydrolysed mainly to tripolyphosphate. It is suggested that the hydrolysis of polyphosphates provides a pH-stat mechanism to counterbalance alkaline stress.

Adenosine Triphosphate↗

PubMed1991

Polyphosphate metabolism in the alga Dunaliella salina studied by 31P-NMR.

Polyphosphate synthesis and the state of the intracellular polyphosphates in the unicellular green alga Dunaliella salina were studied using in vivo 31P-NMR spectroscopy. By perfusing phosphate-depleted algal cells trapped inside agarose beads with orthophosphate (Pi) containing medium, we were able to follow the process of polyphosphate synthesis in whole, living cells. The results suggest that, in Dunaliella, low molecular weight, probably cyclic, polyphosphate intermediates are synthesized from Pi, and are then condensed to high molecular weight polymers. Studies of the intracellular organization of the polyphosphates by electron microscopy and solid-state NMR techniques indicate that most of these polymers are stored in the cell in a soluble form, and not in solid-like structures.

Chlorophyta↗

PubMed1988

Utilization by Escherichia coli of a high-molecular-weight, linear polyphosphate: roles of phosphatases and pore proteins.

We observed that wild-type Escherichia coli utilized a linear polyphosphate with a chain length of 100 phosphate residues (poly-P100) as the sole source of phosphate in growth medium. A mutation in the gene phoA of alkaline phosphatase or phoB, the positive regulatory gene, prevented growth in this medium. Since no alkaline phosphatase activity was detected outside the wild-type cells, the periplasmic presence of the enzyme was necessary for the degradation of polyphosphate. A 90% reduction in the activity of periplasmic acid phosphatase with a pH optimum of 2.5 (delta appA mutants) did not affect polyphosphate utilization. Of the porins analyzed (OmpC, OmpF, and PhoE), the phoB-inducible porin PhoE was not essential since its absence did not prevent growth. To study how poly-P100 diffused into the cells, we used high-resolution 31P nuclear magnetic resonance (31P NMR) spectroscopy. The results suggest that poly-P100 entered the periplasm and remained in equilibrium between the periplasm and the medium. When present individually, porins PhoE and OmpF facilitated a higher permeability for poly-P100 than porin OmpC did. The degradation of polyphosphate by intact cells of E. coli observed by 31P NMR showed a time-dependent increase in cellular phosphate and a decrease in polyphosphate concentration.

Alkaline Phosphatase↗

PubMed1991

Role for ionotropic and metabotropic receptors in quisqualate-stimulated inositol polyphosphate accumulation in rat cerebral cortex.

The actions of the excitatory amino acid quisqualate (QA) on inositol polyphosphate accumulation in cerebral cortex slices have been assessed using both [3H]inositol prelabeling and mass measurements over relatively short incubation periods. QA stimulated accumulation of all the inositol polyphosphates, with similar EC50 values (2.8 +/- 0.7 microM). High performance liquid chromatography analysis of isomeric forms of inositol polyphosphates and specific mass assays revealed that both phosphorylation and dephosphorylation products of inositol-1,4,5-trisphosphate accumulate. A large component of the QA-stimulated inositol polyphosphate accumulation was inhibited by the ionotropic antagonist 6,7-dinitroquinoxaline-2,3-dione in a competitive manner. This implied that the QA response may be due to entry of Ca2+ via voltage-sensitive calcium channels as a consequence of an ionotropic receptor-induced depolarization. In support of this mechanism, the QA-induced response was dependent on the presence of extracellular calcium, whereas the well characterized muscarinic receptor agonist response to carbachol showed only a slight reduction under the same conditions. The concentration-dependent (EC50 8.8 +/- 3 microM) response to the selective ionotropic agonist amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) differed from that to QA or carbachol, in that accumulation of only [3H]inositol mono- and bisphosphates was stimulated, with no increase in the [3H]inositol tris- or tetrakisphosphates. Use of the metabotropic agonist (trans)-(+/-)-1-aminocyclopentyl-1,3-dicarboxylate (ACPD), however, produced concentration-dependent increases in all [3H]inositol polyphosphates. Although both AMPA and ACPD responses alone were smaller in magnitude than that to QA, when present together AMPA and ACPD produced additive responses on [3H]inositol mono- and bisphosphate and a marked synergistic increase in [3H]inositol tetrakisphosphate accumulation, resulting in a response similar to that seen for QA. These data suggest that QA-evoked responses in rat cortex slices are the result of a complex interaction mediated through both ionotropic and metabotropic receptors, in which Ca2+ entry may stimulate accumulation of inositol mono- and bisphosphate directly and divert the metabolism of inositol-1,4,5-trisphosphate to inositol-1,3,4,5-tetrakisphosphate.

Animals↗

PubMed1990

The isolation and characterization of inositol polyphosphate 4-phosphatase.

We previously identified an alternative pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) in calf brain. The enzyme responsible for the degradation of Ins(1,3,4)P3 was designated as inositol polyphosphate 4-phosphatase (Bansal, V. S., Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 9644-9647). We have now purified this enzyme 3390-fold from calf brain-soluble fraction. The isolated enzyme has an apparent molecular mass of 110 kDa as determined by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme migrates as a protein of 105 kDa, suggesting that it is monomeric. Among various 4-phosphate-containing inositol polyphosphates, the enzyme hydrolyzes only Ins(1,3,4)P3 and inositol 3,4-bisphosphate (Ins(3,4)P2), yielding inositol 1,3-bisphosphate and inositol 3-phosphate as products. The inositol polyphosphate 4-phosphatase has apparent Km values of 40 and 25 microM for Ins(1,3,4)P3 and Ins(3,4)P2, respectively. The maximum velocities for these two substrates are 15-20 mumol of product/min/mg protein. Ins(1,3,4)P3 is a competitive inhibitor of Ins(3,4)P2 hydrolysis with an apparent Ki of 27 microM implying that the same active site is involved in hydrolysis of both substrates. The final enzyme preparation retained a small inositol polyphosphate 3-phosphatase activity (less than 2% of rate of inositol polyphosphate 4-phosphatase activity) which most likely reflects a contaminant. The enzyme displays maximum activity between pH 6.5 and 7.5. It is not inhibited by Li+, Ca2+, or Mg2+ except at 10 mM divalent ions. Mn2+ inhibits enzyme at high concentrations IC50 = 1.5 mM.

Animals↗

PubMed1989

Influence of environmental parameters on polyphosphate accumulation in Acinetobacter sp.

The regulation of and the optimum conditions for polyphosphate accumulation in Acinetobacter sp. were determined. Acinetobacter strain 210A accumulated polyphosphate in the presence of an intra- or extracellular energy source. The accumulation of polyphosphate during endogenous respiration was stimulated by streptomycin and inhibited by KCN. The highest amount of polyphosphate was found in cells in which energy supply was not limited, namely at low growth rates under sulphur limitation, and in the stationary phase of growth when either the nitrogen or the sulphur source was depleted. The phosphorus accumulation was not affected by the pH between 6.5 and 9. There was a pronounced effect of the temperature on phosphorus accumulation but is varied from strain to strain. Acinetobacter strain 210A accumulated more phosphate at low temperatures, strain B8 showed an optimum accumulation at 27.5 degrees C, while strain P accumulated phosphorus independently of the temperature. The optimum temperature for growth of Acinetobacter strains tested ranged from 25 to 33 degrees C, and the optimum pH was between 6 and 9.

Acinetobacter↗

PubMed1989

Partial purification of inositol polyphosphate 1-phosphomonoesterase with characterization of its substrates and products by nuclear magnetic resonance spectroscopy.

A study of the enzyme activities that degrade Ins(1,3,4)P3 in rat brain showed that it was dephosphorylated primarily by a Mg2+-dependent inositol polyphosphate 1-phosphomonoesterase to Ins(3,4)P2 and then to Ins(3)P by a 4-phosphomonoesterase. A less active enzyme activity with the properties of a 4-phosphomonoesterase that converted Ins(1,3,4)P3 to Ins(1,3)P2 was also detected. The inositol polyphosphate 1-phosphomonoesterase was separated from the 4-phosphomonoesterase and the inositol monophosphate phosphomonoesterase by chromatography on phosphocellulose, DE-52 anion exchange and hydroxylapatite columns. Kinetic characterization of the partially purified inositol polyphosphate 1-phosphomonoesterase indicated that both Ins(1,3,4)P3 and Ins(1,4)P2 were substrates with apparent Km values of 0.9 microM and 0.7 microM, respectively. Either substrate was a competitive inhibitor of the other substrate and dephosphorylation of both substrates was directly inhibited by Li+ in an uncompetitive manner. These data strongly suggest that a single enzyme dephosphorylates both Ins(1,3,4)P3 and Ins(1,4)P2. The 4-phosphomonoesterase that dephosphorylated Ins(3,4)P2 to Ins(3)P was insensitive to Mg2+ and Li+ and was probably the same enzyme that degraded Ins(1,3,4)P3 to Ins(1,3)P2. The isomeric configurations of the major inositol polyphosphates formed from the degradation of Ins(1,3,4,5)P4 were determined using 1H- and 31P-NMR spectroscopy, and confirmation of the structures assigned to Ins(1,3,4,5)P4, Ins(1,3,4)P3 and Ins(3,4)P2 was obtained.

Animals↗

PubMed1990

Polyphosphate production by strains of Acinetobacter.

Of four strains of Acinetobacter isolated from a pilot plant exhibiting enhanced biological phosphate removal from sewage, two strains (RA3116 and RA3117) accumulated more than 10 times the amount of polyphosphate accumulated by the other two strains (RA3114 and RA3123). Variants isolated from RA3116 and RA3117 showed polyphosphate levels similar to RA3114 and RA3123. No correlation was found between the polyphosphate content of the strains and levels of several enzymes that have been implicated in polyphosphate formation.

Acinetobacter↗

PubMed1978

Polyphosphate-deficient mutants of Anacystis nidulans.

Polyphosphate-deficient mutants of Anacystis nidulans have been isolated by either ethyl methanesulfonate (EMS) or N-methyl nitrosoguanidine (NTG) mutagenesis and penicillin-enrichment techniques. Mutagenised stock was preincubated in a medium lacking sulfate, then transferred to a phosphate-lacking medium before penicillin treatment. Many single-colony isolates, in contrast to wild-type, show little growth in absence of phosphate, and have altered polyphosphate, and have altered polyphosphate kinase levels indicating that the lesions affect either the activity or the expression of this enzyme. In these same mutants radioactive phosphate incorporation is severely retarded. Electron micrographs confirm the absence of polyphosphate granules in some mutants.

Cyanobacteria↗

PubMed1978

[Inorganic polyphosphate metabolism in Staphylococcus aureus and the action on it of antibiotics].

The culure of Staph. aureus in the exponential growth phase contained 14-18 mg/g of dry orthophosphate biomass and 18-22 mg/g of dry acid insoluble polyphosphate biomass. The extracellular extract of the culture had a phosphohydrolase activity with respect to high molecular polyphosphates, tripolyphosphate and pyrophosphate. Penicillin and bacitracin which inhibited the biosynthesis of the cell wall had no effect on the content of polyphosphates and the phosphohydrolase activity of Staph. aureus. Heliomycin which inhibited the biosynthesis of RNA increased the content of polyphosphates by 1.5 times and decreased the content of ATP by 30 per cent in the cells of Staph. aureus.

Adenosine Triphosphate↗

PubMed1991

Formation of inositol polyphosphates in cultured adrenal chromaffin cells.

Formation of inositol polyphosphates has been characterized in cultured bovine adrenal chromaffin cells in terms of calcium dependency and isomers of inositol polyphosphates. There are two distinct pathways of generation of InsP3. Stimulants such as high K+ induce InsP3 accumulation by a calcium uptake-dependent mechanism. Stimulants such as Ang II induce InsP3 accumulation by a calcium uptake-independent mechanism. Both mechanisms are involved in nicotinic stimulation. These results suggest that calcium entry as well as receptor-mediated mechanisms play a significant role in phosphoinositides hydrolysis through phospholipase C in adrenal chromaffin cells. Nicotinic receptor stimulation induces a rapid and transient increase in Ins(1,4,5)P3 accumulation followed by a slower accumulation of Ins(1,3,4)P3. Moreover, nicotine induces a large and rapid increase in Ins(1,3,4,5,6)P5 accumulation with an extent and time course similar to Ins(1,4,5)P3, which peaks at 15 sec after stimulation. Nicotine also induced Ins(1,3,4,5)P4 and InsP6 accumulation with a slower time course and a lesser magnitude than Ins(1,3,4,5,6)P5. These results indicate that adrenal chromaffin cells possess fine regulation of inositol polyphosphates metabolism and that inositol polyphosphates are involved with the control of cellular function in these cells.

Adrenal Medulla↗

PubMed1976

Role of ATP-glucokinase and polyphosphate glucokinase in Streptomyces aureofaciens.

The activity of ATP-glucokinase and of polyphosphate glucokinase was examined during growth of the actinomycete Streptomyces aureofaciens 8425 under conditions of intense chlortetracycline (CTC) synthesis. ATP-glucokinase was active in the strain only during the logarithmic phase of culture growth; the activity of polyphosphate glucokinase appears only at the end of the logarithmic phase of growth and rises in parallel with the rate of CTC biosynthesis in the stationary phase. During the rise of activity of polyphosphate glucokinase and of CTC biosynthesis the cells accumulate sugar phosphates, mainly glucose-6-phosphate. It appears that the biosynthesis of CTC in Streptomyces aureofaciens takes place at the expense of glycolysis, using up the high-energy phosphate of high-molecular polyphosphates.

Adenosine Triphosphate↗

PubMed1987

Effects of growth state and amines on cytoplasmic and vacuolar pH, phosphate and polyphosphate levels in Saccharomyces cerevisiae: a 31P-nuclear magnetic resonance study.

The vacuoles of logarithmic and stationary stage cells were compared by 31P-NMR with regard to pH, orthophosphate (Pi) content and average size of polyphosphate. The vacuoles of stationary cells had lower pH, higher Pi content, and polyphosphates of longer average chain length, although total polyphosphate content was about the same as in logarithmic cells. The lower vacuolar pH in stationary cells was the major cause of a larger cytoplasmic-vacuolar pH gradient. Addition of NH4Cl, (NH4)2SO4, methylamine or amantadine at pH 8 to cells in either stage caused an increase in both cytoplasmic and vacuolar pH, with little or no change in the cytoplasmic-vacuolar pH gradient. However, the administration of ammonium salts to the cells at pH 8.0 resulted in rapid hydrolysis of the intravacuolar polyphosphate to tripolyphosphate and Pi, with attendant redistribution of Pi between the vacuolar and cytoplasmic compartments.

Amantadine↗