Properties of polyphosphatase ofAcinetobacter johnsonii 210A

Polyphosphatase, an enzyme which hydrolyses highly polymeric polyphosphates to P_i, was purified 77-fold fromAcinetobacter johnsonii 210A by Q-Sepharose, hydroxylapatite and Mono-Q column chromatography. The native molecular mass estimated by gel filtration and native gel electrophoresis was 55 kDa. SDS-polyacrylamide gel electrophoresis indicated that polyphosphatase ofAcinetobacter johnsonii 210A is a monomer. The enzyme was specific for highly polymeric polyphosphates and showed no activity towards pyrophosphate and organic phosphate esters. The enzyme was inhibited by iodoacetamide and in the presence of 10 mM Mg²⁺ by pyro- and triphosphate. The apparent K_m-value for polyphosphate with an average chain length of 64 residues was 5.9 µM and for tetraphosphate 1.2 mM. Polyphosphate chains were degraded to short chain polymers by a processive mechanism. Polyphosphatase activity was maximal in the presence of Mg²⁺ and K⁺.     

Partial Purification and Characterization of Nuclear Exopolyphosphatase from Saccharomyces cerevisiae Strain with Inactivated PPX1 Gene Encoding a Major Yeast Exopolyphosphatase

Inactivation of PPX1 encoding the major cytosolic exopolyphosphatase PPX1 in Saccharomyces cerevisiae did not alter exopolyphosphatase activity of the isolated nuclei compared with that in the parent strain. The nuclear exopolyphosphatase of the S. cerevisiae strain deficient in the PPX1 gene was purified 10-fold. According to gel filtration on Superose 6, this enzyme has a molecular mass of approximately 200 kD, and it hydrolyzes polyphosphates with an average chain length of 15 and 208 phosphate residues to the same extent. Its activity is much lower with tripolyphosphate. In the presence of 2.5 mM Mg2+, Km values are 133 and 25 microM in the hydrolysis of polyphosphates with chain lengths of 15 and 208 phosphate residues, respectively. The enzyme activity is stimulated by 2.5 mM Mg2+ and 0.1 mM Co2+ 15- and 31-fold, respectively. RNA does not alter the nuclear exopolyphosphatase activity, while polylysine increases it 2-fold.     

Purification and characterization of a soluble polyphosphatase from mitochondria of Saccharomyces cerevisiae

A polyphosphatase with the specific activity 2.2 U/mg was purified to apparent homogeneity from a soluble preparation of mitochondria of Saccharomyces cerevisiae. The polyphosphatase is a monomeric protein of approximately 41 kD. The purified enzyme hydrolyzes polyphosphates with an average chain length of 9 to 208 phosphate residues to the same extent, but its activity is approximately 2-fold higher with tripolyphosphate. ATP, PPi, and p-nitrophenyl phosphate are not substrates of this enzyme. The apparent Km values are 300, 18, and 0.25 microM obtained at hydrolysis of polyphosphates with a chain length of 3, 15, and 188 phosphate residues, respectively. Several divalent cations stimulated the enzyme activity 1.2-27-fold (Mg2+ = Co2+ = Mn2+ > Zn2+). Determination of the protein N-terminal sequence and its comparison with the EMBL data library indicates that the soluble polyphosphatase of mitochondria of S. cerevisiae is not encoded by the gene of the major yeast polyphosphatase PPX1.     

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.     

Inorganic polyphosphates in mitochondria

Current data concerning the crucial role of inorganic polyphosphates (polyP) in mitochondrial functions and dysfunctions in yeast and animal cells are reviewed. Biopolymers with short chain length (∼15 phosphate residues) were found in the mitochondria of Saccharomyces cerevisiae. They comprised 7–10% of the total polyP content of the cell. The polyP are located in the membranes and intermembrane space of mitochondria. The mitochondrial membranes possess polyP/Ca²⁺/polyhydroxybutyrate complexes. PolyP accumulation is typical of promitochondria but not of functionally active mitochondria. Yeast mitochondria possess two exopolyphosphatases splitting P_i from the end of the polyP chain. One of them, encoded by the PPX1 gene, is located in the matrix; the other one, encoded by the PPN1 gene, is membrane-bound. Formation of well-developed mitochondria in the cells of S. cerevisiae after glucose depletion is accompanied by decrease in the polyP level and the chain length. In PPN1 mutants, the polyP chain length increased under glucose consumption, and the formation of well-developed mitochondria was blocked. These mutants were defective in respiration functions and consumption of oxidizable carbon sources such as lactate and ethanol. Since polyP is a compound with high-energy bonds, its metabolism vitally depends on the cell bioenergetics. The maximal level of short-chain acid-soluble polyP was observed in S. cerevisiae under consumption of glucose, while the long-chain polyP prevailed under ethanol consumption. In insects, polyP in the mitochondria change drastically during ontogenetic development, indicating involvement of the polymers in the regulation of mitochondrial metabolism during ontogenesis. In human cell lines, specific reduction of mitochondrial polyP under expression of yeast exopolyphosphatase PPX1 significantly modulates mitochondrial bioenergetics and transport.     

Inorganic polyphosphate in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with a mutation disturbing the function of vacuolar ATPase

A mutation in the vma2 gene disturbing V-ATPase function in the yeast Saccharomyces cerevisiae results in a five- and threefold decrease in inorganic polyphosphate content in the stationary and active phases of growth on glucose, respectively. The average polyphosphate chain length in the mutant cells is decreased. The mutation does not prevent polyphosphate utilization during cultivation in a phosphate-deficient medium and recovery of its level on reinoculation in complete medium after phosphate deficiency. The content of short chain acid-soluble polyphosphates is recovered first. It is supposed that these polyphosphates are less dependent on the electrochemical gradient on the vacuolar membrane.     

Teichulosonic acid,an anionic polymer of a new class from the cell wall of <Emphasis Type="Italic">Actinoplanes utahensis</Emphasis> VKM Ac-674<Superscript>T</Superscript>

The cell wall of Actinoplanes utahensis VKM Ac-674^T contains two anionic polymers: teichoic acid 1,3-poly(glycerol phosphate) that is widespread in cell walls of Gram-positive bacteria; and a unique teichulosonic acid belonging to a new class of bioglycans described only in microorganisms of the Actinomycetales order. The latter polymer contains residues of di-N-acyl derivative of sialic acid-like monosaccharide — 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-β-L-manno-non-2-ulosonic or pseudaminic acid (Pse) which bears the N-(3,4-dihydroxybutanoyl) group (Dhb) at C7. This polymer has irregular structure and consists of fragments of two types, which differ in substitution of the Dhb residues at O4 either with β-D-glucopyranose or with β-Pse residues. Most of the β-Pse residues (∼80%) are glycosylated at position 4 with α-D-galactopyranose residues in both types of fragments. The glucose, galactose, and Dhb residues are partly O-acetylated. The structures of the polymers were established by chemical and NMR spectroscopy methods.     

Effects of inactivation of the PPN1 gene on exopolyphosphatases, inorganic polyphosphates and function of mitochondria in the yeast Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae with inactivated endopolyphosphatase gene PPN1 did not grow on lactate and ethanol, and stopped growth on glucose earlier than the parent strain. Their mitochondria were defective in respiration functions and in metabolism of inorganic polyphosphates. The PPN1 mutants lacked exopolyphosphatase activity and possessed a double level of inorganic polyphosphates in mitochondria. The average chain length of mitochondrial polyphosphates at the stationary growth stage on glucose was about 15-20 and about 130-180 phosphate residues in the parent strain and PPN1 mutants, respectively. Inactivation of the PPX1 gene encoding exopolyphosphatase had no effect on respiration functions and on polyphosphate level and chain length in mitochondria.     

Accumulation of phosphate and polyphosphate by Cryptococcus humicola and Saccharomyces cerevisiae in the absence of nitrogen

The search for new phosphate-accumulating microorganisms is of interest in connection with the problem of excess phosphate in environment. The ability of some yeast species belonging to ascomycetes and basidiomycetes for phosphate (P (i) ) accumulation in nitrogen-deficient medium was studied. The ascomycetous Saccharomyces cerevisiae and Kuraishia capsulata and basidiomycetous Cryptococcus humicola, Cryptococcus curvatus, and Pseudozyma fusiformata were the best in P (i) removal. The cells of Cryptococcus humicola and S.?cerevisiae took up 40% P (i) from the media containing P (i) and glucose (5 and 30?mM, respectively), and up to 80% upon addition of 5?mM MgSO(4) (.) The cells accumulated P (i) mostly in the form of polyphosphate (PolyP). In the presence of Mg(2+) , the content of PolyP with longer average chain length increased in both yeasts; they both had numerous inclusions fluorescing in the yellow region of the spectrum, typical of DAPI-PolyP complexes. Among the yeast species tested, Cryptococcus humicola is a new promising model organisms to study phosphorus removal from the media and biomineralization in microbial cells.     

Rat polymerase beta gapped DNA interactions: antagonistic effects of the 5' terminal PO4 - group and magnesium on the enzyme binding to the gapped DNAs with different ssDNA gaps

The role of the 5′ terminal phosphate group downstream from the primer and magnesium cations in the energetics and dynamics of the gapped DNA recognition by rat polymerase β have been examined, using the fluorescence titration and stopped-flow techniques. The analyses have been performed with the entire series of gapped DNA substrates differing in the size of the ssDNA gap. The 5′ terminal phosphate group and magnesium cations exert antagonistic effect on enzyme binding to gapped DNA that depends on the length of the ssDNA gap. The PO ₄ ⁻ group amplifies the differences between the substrates with different ssDNA gaps, while in the presence of magnesium, affinities and structural changes induced in the DNA are very similar among examined DNA substrates. Both, the phosphate group and Mg⁺² differ dramatically in affecting the thermodynamic response of the gapped DNA-rat pol β system to the salt concentration. The data indicate that these distinct effects result from affecting the structure of the DNA, in the case of the phosphate group, and from direct magnesium binding to the protein. The mechanism of rat enzyme binding depends on the length of the ssDNA gap and the presence of the 5′ terminal phosphate group. Complex formation with DNAs having three, four, and five residues in the gap occurs by a minimum three-step sequential mechanism. Depending on the presence of the 5′ terminal phosphate group and/or magnesium, binding of the enzyme to a DNA containing two residues in the ssDNA gap is described by the same three-step or by a simpler two-step mechanism. With the DNA containing only one residue in the gap, binding is always described by only a two-step mechanism. The PO ₄ ⁻ group and magnesium cations have opposite effects on internal stability of the complexes with different length of the ssDNA gap. While the PO ₄ ⁻ group increases the stability of internal intermediates with the increasing length of the gap, Mg⁺² decreases the stability of the intermediates with longer ssDNA gap. As a result, the combined favorable orientation effect of the phosphate group and the unfavorable Mg⁺² effect lead to the optimal docking of the ssDNA gaps with three and four residues by the enzyme. This work was supported by NIH Grant GM-58565 (to W. B.)     

Ion Content of the Halotolerant Alga Dunaliella salina

The intracellular concentration of the major ions in Dunaliellasalina cells were determined, following the removal of extracellularions by ion-exchange minicolumns. Log phase cells, grown inmedia containing 1–4 molar NaCl, contained 30–50mM chloride and 200–350 mM magnesium (5 mM in medium).Phosphorus, which is present intracellularly mostly as polyphosphate,was present in amounts of 60–100 fmoles per cell, equivalentto a concentration of 600–1,000 mM (0.2 mM in medium).Previous data indicated that such cells contained 20–40mM Na⁺, 150–300mM K⁺, 20mM SO^2–₄, and very low concentrationsof Ca2⁺ and charged nitrogenous compounds. Mg²⁺ and K⁺ seemto serve as the major counter ions for the intracellular negativecharge present in the massively accumulated polyphosphates.The former accounts for about 2/3 of the required positive charge.This is supported by the observation that limitation in thephosphate or K⁺ supply in the medium lead to a parallel decreasein the accumulation of intracellular phosphorus, Mg²⁺ or K⁺. ¹Present address: Department of Vegetables, The Volcani Center,Bet-Dagan 50250, Israel. (Received June 13, 1988; Accepted August 25, 1988)     

Subcellular Distribution of Inorganic Phosphate, and Levels of Nucleoside Triphosphate, in Mature Maize Roots at Low External Phosphate Concentrations: Measurements with 31P-NMR

Maize plants were grown in nutrient solution without phosphate,or in which inorganic phosphate (Pi) was maintained at nearlyconstant concentrations of 1 µM, 10µM or 0·5mM. In vivo ³¹P-NMR measurements showed that there was no discernibledifference in the cytoplasmic Pi content (µmol cm^–3root volume) of the mature roots of plants exposed to 1 µM,10µM or 0·5 mM external phosphate for up to 12d. However, the vacuolar Pi content of the mature roots variedabout 10-fold between these three groups. The cytoplasmic Pi content of roots receiving no external phosphatedecreased significantly after about 7 d total growth, and atabout this time the vacuolar pool of Pi became too small foraccurate measurement. The presence of 1 µM Pi in the nutrientsolution completely prevented this decline in cytoplasmic Pi,and there was some evidence that it also raised the Pi contentof the root vacuoles above the almost undetectable level foundin the totally P-starved roots. During the first 7–9 d of growth, the nucleoside triphosphatecontent of the mature roots was unaffected by the concentrationof phosphate in the nutrient solution. The results highlight the close control of cytoplasmic concentrationsof certain important phosphorus metabolites in roots growingin soil of normal agricultural fertility. Key words: Vacuole, cytoplasm, intracellular compartmentation, NTP, P-nutrition     

Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess

Isolated mitochondria of Saccharomyces cerevisiae cells grown on glucose possess acid-soluble inorganic polyphosphate (polyP). Its level strongly depends on phosphate (P(i)) concentration in the culture medium. The polyP level in mitochondria showed 11-fold decrease under 0.8 mM P(i) as compared with 19.3 mM P(i). When spheroplasts isolated from P(i)-starved cells were incubated in the P(i)-complete medium, they accumulated polyP and exhibited a phosphate overplus effect. Under phosphate overplus the polyP level in mitochondria was two times higher than in the complete medium without preliminary P(i) starvation. The average chain length of polyP in mitochondria was of <15 phosphate residues at 19.3 mM P(i) in the culture medium and increased at phosphate overplus. Deoxyglucose inhibited polyP accumulation in spheroplasts, but had no effect on polyP accumulation in mitochondria. Uncouplers (FCCP, dinitrophenol) and ionophores (monensin, nigericin) inhibited polyP accumulation in mitochondria more efficiently than in spheroplasts. Fast hydrolysis of polyP was observed after sonication of isolated mitochondria. Probably, the accumulation of polyP in mitochondria depended on the proton-motive force of their membranes.     

A 31P nuclear magnetic resonance study of phosphate levels in roots of ectomycorrhizal and nonmycorrhizal plants of Castanea sativa Mill.

 ³¹P-Nuclear Magnetic Resonance (NMR) was used to assess phosphate distribution in ectomycorrhizal and nonmycorrhizal roots of Castanea sativa Mill. as well as in the mycorrhizal fungus Pisolithus tinctorius in order to gain insight into phosphate trafficking in these systems. The fungus P. tinctorius accumulated high levels of polyphosphates during the rapid phase of growth. Mycorrhizal and nonmycorrhizal roots accumulate orthophosphate. Only mycorrhizal roots presented polyphosphates. The content in polyphosphates increased along the 3 months of mycorrhiza formation. In mycorrhizal roots of plants cultured under axenic conditions, the orthophosphate pool decreased along the culture time. In nonmycorrhizal roots the decrease in the orthophosphate content was less pronounced. The level of orthophosphate in mycorrhizal roots was significantly lower than in nonmycorrhizal ones, which indicates that this system relies upon the fungal polyphosphates as a major source of phosphate. Received: 28 July 1998 / Accepted: 21 October 1998     

Phosphate accumulation of <Emphasis Type="Italic">Acetobacter xylinum</Emphasis>

The cells of Acetobacter xylinum decreased phosphate concentration in the medium from 5 to 2.5 or 0.3 mM during incubation in the presence of Mg²⁺ and glucose, or Mg²⁺ and casamino acids, respectively. The prevalence of orthophosphate or polyphosphate in the biomass of A. xylinum depends on the medium composition. Under phosphate uptake in the presence of glucose, the content of orthophosphate in the biomass changed little, while that of polyphosphate increased fourfold. At incubation with casamino acids, the content of orthophosphate increased 15 times, while that of polyphosphate increased only 2.5 times. Some part of orthophosphate in this case seems to be bound with the cell surface. The polyphosphate chain length in the cells of A. xylinim increases under phosphate uptake. This increase is more noticeable in the presence of glucose. Casamino acids can be replaced by α-ketoglutaric acid in combination with (NH₄)₂SO₄, or arginine, or glutamine, the catabolism of which results in formation of NH₄ ⁺ and α-ketoglutarate.     

Heavy metals and thiol pool in three strains of <Emphasis Type="Italic">Tetracladium marchalianum</Emphasis>

Growth of three strains of Tetracladium marchalianum was inhibited by Cd-, and, to a lesser extent, by Cu-and Zn-chloride. In the presence of 50 μM Cd(II), all strains increased total thiol and glutathione production to 6, 11, and 21 μmoles · mg⁻¹ dry mass, respectively. Cd(II) also induced the synthesis of one to several compounds reacting with 5,5′-dithio-bis-(2-nitrobenzoic acid). In order to identify buffer-soluble thiolic compounds other than cysteine, γ-EC and γ-ECG (glutathione) were analyzed and confirmed by mass spectrometry. No water soluble sulfides were detectable in any of the culture filtrates, but Cd(II) exposure at a concentration of 50 μM raised sulfide levels in the mycelia of two of the strains between 3 and 7-fold, Cu(II) and Zn(II) had no effect. Energy Dispersive X-ray-analysis (EDX) and Electron Spectrometry-Images (ES-I) of one strain revealed increased levels of Cu and Zn in the cytoplasm and even higher levels in vacuolar precipitates. Zn and Cu are accumulated in the vacuoles as polyphosphates, identified by Electron Energy Loss-Spectrometry (EELS). Cd was found only in the vacuoles.     

Interrelationship of polyphosphate metabolism and levorin biosynthesis in Streptomyces levoris

The effect of inorganic phosphate on biosynthesis of the polyene antibiotic levorin by Streptomyces levoris was studied. At phosphate concentration of 4.0 mM levorin biosynthesis is repressed by 90%, resulting in an increase of ATP and a condensed inorganic polyphosphates content in the producer cells. At phosphate concentration optimal for levorin production (0.04 mM) the level of intracellular ATP sharply falls by the beginning of the steady-state phase of the producer growth and that of polyphosphates decreases 3-6-fold. The inorganic phosphate exerts different effects on polyphosphate metabolism enzymes, such as polyphosphate: D-glucose-6-phosphotransferase, polyphosphate phosphohydrolase, tripolyphosphate phosphohydrolase, pyrophosphate phosphohydrolase, alkaline and acid phosphatase. The strongest effect of phosphate excess is observed in the case of polyphosphate: D-glucose-6-phosphotransferase, whose activity decreases 2-5-fold. The activity of this enzyme was shown to be correlated with the antibiotic accumulation. The data obtained are indicative of interrelationship between the polyphosphate metabolism and levorin biosynthesis.     

Formation of insoluble magnesium phosphates during growth of the archaea Halorubrum distributum and Halobacterium salinarium and the bacterium Brevibacterium antiquum

Stationary phase cells of the halophilic archaea Halobacterium salinarium and Halorubrum distributum, growing at 3-4 M NaCl, and of the halotolerant bacterium Brevibacterium antiquum, growing with and without 2.6 NaCl, took up approximately 90% of the phosphate from the culture media containing 2.3 and 11.5 mM phosphate. The uptake was blocked by the uncoupler FCCP. In B. antiquum, EDTA inhibited the phosphate uptake. The content of polyphosphates in the cells was significantly lower than the content of orthophosphate. At a high phosphate concentration, up to 80% of the phosphate taken up from the culture medium was accumulated as Mg(2)PO(4)OH x 4H(2)O in H. salinarium and H. distributum and as NH(4)MgPO(4) x 6H(2)O in B. antiquum. Consolidation of the cytoplasm and enlargement of the nucleoid zone were observed in the cells during phosphate accumulation. At phosphate surplus, part of the H. salinarium and H. distributum cell population was lysed. The cells of B. antiquum were not lysed and phosphate crystals were observed in the cytoplasm.     

Phosphate-dependent glutaminase in enterocyte mitochondria and its regulation by ammonium and other ions

Summary.  The effects of ammonium and other ions on phosphate dependent glutaminase (PDG) activity in intact rat enterocyte mitochondria were investigated. Sulphate and bicarbonate activated the enzyme in absence and presence of added phosphate. In presence of 10 mM phosphate, ammonium at concentrations <1 mM inhibited the enzyme. This inhibition was reversed by increased concentration of phosphate or sulphate. The inhibition of PDG by ammonium in presence of 10 mM phosphate was biphasic with respect to glutamine concentration, its effect being through a lowering of V_max at glutamine concentration of ≤5 mM, and increased K_m for substrate concentration above 5 mM. The activation of the enzyme by bicarbonate was through an increase in V_max. Ammonium and bicarbonate ions may therefore be important physiological regulators of PDG. It is suggested that phosphate and other polyvalent ions may function by preventing product inhibition of the enzyme through promotion of PDG dimer formation. The dimerized enzyme may have a high affinity for glutamine and reduced sensitivity to inhibition by ammonium ions. Received August 10, 2001 Accepted April 1, 2002 Published online August 30, 2002 Acknowledgement This work was supported by University of Zimbabwe research grant to Dr. B. Masola. Authors' address: Dr. Bubuya Masola, Department of Biochemistry, University of Zimbabwe, P O Box MP167, Mount Pleasant, Harare, Zimbabwe, E-mail: masolab@yahoo.co.uk