Tatiana Kulakovskaya

New aspects of inorganic polyphosphate metabolism and function.

The review analyzes the results of recent studies on the biochemistry of high-molecular inorganic poly-phosphates (PolyPs). The data obtained lead to the following main conclusions. PolyPs are polyfunctional compounds. The main role of PolyPs is their participation in the regulation of metabolism both at the genetic and metabolic levels. Among the functions of PolyPs known at present, the most important are the following: phosphate and energy storage; regulation of the levels of ATP and other nucleotide and nucleoside-containing coenzymes; participation in the regulation of homeostasis and storage of inorganic cations and other positively charged solutes in an osmotically inert form; participation in membrane transport processes mediated by poly-beta-Ca2+-hydroxybutyrate complexes; participation in the formation and functions of cell surface structures; control of gene activity; and regulation of activities of the enzymes and enzyme assemblies involved in the metabolism of nucleic acids and other acid biopolymers. However, the functions of PolyPs vary among organisms of different evolutionary levels. The metabolism and functions of PolyPs in each cellular compartment of procaryotes (cell wall, plasma membrane, cytosol) and eucaryotes (nuclei, vacuoles, mitochondria, plasma membrane, cell wall, mitochondria, cytosol) are unique. The synthesis and degradation of PolyPs in the organelles of eucaryotic cells are possibly mediated by different sets of enzymes. This is consistent with of the endosymbiotic hypothesis of eucaryotic cell origin. Some aspects of the biochemistry of high-molecular PolyPs are considered to be of great significance to the approach to biotechnological, ecological and medical problems.

ATP leakage from yeast cells treated by extracellular glycolipids of Pseudozyma fusiformata

The ustilaginaceous yeast Pseudozyma fusiformata secreted glycolipids which were lethal to many yeasts and fungi more active at pH of about 4.0, and in the temperature range of 20-30 degrees C. Purified glycolipids enhanced non-specific permeability of the cytoplasmic membrane in sensitive cells, which resulted in ATP leakage and susceptibility of the cells to staining with bromocresol purple. Cells of Saccharomyces cerevisiae lost the ability to acidify the medium. Basidiomycetous yeasts were more sensitive to the glycolipids than ascomycetous ones. The minimal effective glycolipid concentration was 0.13 and 0.26 mg ml(-1) for Cryptococcus terreus and Filobasidiella neoformans, while for Candida albicans and Saccharomyces cerevisiae it was 1.0 and 1.6 mg ml(-1).

Effect of a carbon source on polyphosphate accumulation in Saccharomyces cerevisiae

The cells of Saccharomyces cerevisiae accumulate inorganic polyphosphate (polyP) when reinoculated on a phosphate-containing medium after phosphorus starvation. Total polyP accumulation was similar at cultivation on both glucose and ethanol. Five separate fractions of polyP: acid-soluble fraction polyP1, salt-soluble fraction polyP2, weakly alkali-soluble fraction polyP3, alkali-soluble fraction polyP4, and polyP5, have been obtained from the cells grown on glucose and ethanol under phosphate overplus. The dynamics of polyP fractions depend on a carbon source. The accumulation rates for fractions polyP2 and polyP4 were independent of the carbon source. The accumulation rates of polyP1 and polyP3 were higher on glucose, while fraction polyP5 accumulated faster on ethanol. As to the maximal polyP levels, they were independent of the carbon source for fractions polyP2, polyP3, and polyP4. The maximal level of fraction polyP1 was higher on glucose than on ethanol, but the level of fraction polyP5 was higher on ethanol. It was assumed that accumulation of separate polyP fractions has a metabolic interrelation with different energy-providing pathways. The polyphosphate nature of fraction polyP5 was demonstrated for the first time by (31)P nuclear magnetic resonance spectroscopy, enzymatic assay, and electrophoresis.

Purification and properties of exopolyphosphatase isolated from Saccharomyces cerevisiae vacuoles

An exopolyphosphatase (polyPase) with a specific activity of 60 U/mg protein has been purified from the vacuolar sap of Saccharomyces cerevisiae. The molecular mass of the intact enzyme was found to be 245 kDa. It is highly specific towards high‐molecular polyphosphates (polyP). The activity with polyP9 is 24% of that with polyP208. The apparent K m for polyP15 and polyP208 hydrolysis is 93 and 2.4 μM, respectively. The enzyme is slightly active with polyP3 and adenosine‐5′‐tetraphosphate, but does not hydrolyze pyrophosphate, ATP, GTP and p‐nitrophenylphosphate. It is stimulated by divalent metal cations. Co2+, the best activator, stimulates it 6‐fold. Antibodies that inhibit the cell envelope and cytosol polyPases of S. cerevisiae have no effect on the vacuolar polyPase. The vacuolar polyPase differs from other yeast polyPases in molecular mass, substrate specificity and effects of activators.

Polyphosphatase PPN1 of Saccharomyces cerevisiae: Switching of Exopolyphosphatase and Endopolyphosphatase Activities

The polyphosphatase PPN1 of Saccharomyces cerevisiae shows an exopolyphosphatase activity splitting phosphate from chain end and an endopolyphosphatase activity fragmenting high molecular inorganic polyphosphates into shorter polymers. We revealed the compounds switching these activities of PPN1. Phosphate release and fragmentation of high molecular polyphosphate prevailed in the presence of Co2+ and Mg2+, respectively. Phosphate release and polyphosphate chain shortening in the presence of Co2+ were inhibited by ADP but not affected by ATP and argininе. The polyphosphate chain shortening in the presence of Mg2+ was activated by ADP and arginine but inhibited by ATP.

Adaptation of Saccharomyces cerevisiae to toxic manganese concentration triggers changes in inorganic polyphosphates

The ability of Saccharomyces cerevisiae to adapt to toxic Mn(2+) concentration (4 mM) after an unusually long lag phase has been demonstrated for the first time. The mutants lacking exopolyphosphatase PPX1 did not change the adaptation time, whereas the mutants lacking exopolyphosphatase PPN1 reduced the lag period compared with the wild-type strains. The cell populations of WT and ΔPPN1 in the stationary phase at cultivation with Mn(2+) contained a substantial number of enlarged cells with a giant vacuole. The adaptation correlated with the triggering of polyphosphate metabolism: the drastic increase in the rate and chain length of acid-soluble polyphosphate. The share of this fraction, which is believed to be localized in the cytoplasm, increased to 76%. Its average chain length increased to 200 phosphate residues compared with 15 at the cultivation in the absence of manganese. DAPI-stained inclusions in the cytoplasm were accumulated in the lag phase during the cultivation with Mn(2+).

Synthesis of magneto-sensitive iron-containing nanoparticles by yeasts

Abstract Industrial production of magneto-sensitive nanoparticles, which can be used in the production of target drug delivery carriers, is a subject of interest for biotechnology and microbiology. Synthesis of these nanoparticles by microorganisms has been described only for bacterial species. At the same time, it is well known that yeasts can form various metal-containing nanoparticles used, for instance, in semiconductors, etc. This paper describes the first results of the biosynthesis of magneto-sensitive nanoparticles by yeasts. The organisms we used—Saccharomyces cerevisiae and Cryptococcus humicola—represented two different genera. Magneto-sensitive nanoparticles were synthesized at room temperature in bench-scale experiments. The study included transmission electron microscopy of the yeast cells and their energy dispersive spectrum analyses and revealed the presence of iron-containing nanoparticles. Both yeast cultures synthesized nanoparticles at high concentrations of dissolved iron. Electron microscopy showed that nanoparticles were associated mainly with the yeast cell wall. Formation of magneto-sensitive nanoparticles was studied under conditions of applied magnetic fields; a possible stimulating role of magnetic field is suggested. On the whole, the paper reports a novel approach to green biosynthesis of magneto-sensitive nanoparticles.

V-ATPase dysfunction suppresses polyphosphate synthesis in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae accumulates the high levels of inorganic polyphosphates (polyPs) performing in the cells numerous functions, including phosphate and energy storage. The effects of vacuolar membrane ATPase (V-ATPase) dysfunction were studied on polyP accumulation under short-term cultivation in the Pi–excess media after Pi starvation. The addition of bafilomycin A1, a specific inhibitor of V-ATPase, to the medium with glucose resulted in strong inhibition of the synthesis of long-chain polyP and in substantial suppression of short-chain polyP. The addition of bafilomycin to the medium with ethanol resulted in decreased accumulation of high-molecular polyP, while the accumulation of low-molecular polyP was not affected. The levels of polyP synthesis in the mutant strain with a deletion in the vma2 gene encoding a V-ATPase subunit were significantly lower than in the parent strain in the media with glucose and with ethanol. The synthesis of the longest chain polyP was not observed in the mutant cells. The synthesis of only the low-polymer acid-soluble polyP fraction occurred in the cells of the mutant strain. However, the level of polyP1 was nearly tenfold lower than compared to the cells of the parent strain. Both bafilomycin A1 and the mutation in vacuolar ATPase subunit vma2 lead to a considerable decrease of cellular polyP accumulation. Thus, the defects in ΔμH+ formation on the vacuolar membrane resulted in the decrease of polyP biosynthesis in S. cerevisiae.

Triterpenoid saponins from the roots of Acanthophyllum gypsophiloides Regel

Summary Two new triterpenoid saponins 1 and 2 were isolated from the methanol extract of the roots of Acanthophyllum gypsophiloides Regel. These saponins have quillaic acid or gypsogenin moieties as an aglycon, and both bear similar sets of two oligosaccharide chains, which are 3-O-linked to the triterpenoid part trisaccharide α-L-Arap-(1→3)-[α-D-Galp-(1→2)]-β-D-GlcpA and pentasaccharide β-D-Xylp-(1→3)-β-D-Xylp-(1→3)-α-L-Rhap-(1→2)-[β-D-Quip-(1→4)]-β-D-Fucp connected through an ester linkage to C-28. The structures of the obtained saponins were elucidated by a combination of mass spectrometry and 2D NMR spectroscopy. A study of acute toxicity, hemolytic, anti-inflammatory, immunoadjuvant and antifungal activity was carried out. Both saponins 1 and 2 were shown to exhibit immunoadjuvant properties within the vaccine composition with keyhole limpet hemocyanin-based immunogen. The availability of saponins 1 and 2 as individual pure compounds from the extract of the roots of A. gypsophiloides makes it a prospective source of immunoactive agents.

The cadmium tolerance in Saccharomyces cerevisiae depends on inorganic polyphosphate

The sensitivity to cadmium (Cd(II)), an important environmental pollutant, was studied in the cells of Saccharomyces cerevisiae strains with genetically altered polyphosphate metabolism. The strains overproducing polyphosphatases PPX1 or PPN1 were more sensitive to Cd(II) than the parent strain. The half maximal inhibitory concentrations were 0.02 and 0.05 mM for the transformants and the parent strain, respectively. Transformant strains cultivated in the presence of Cd(II) show a decrease in the content of short‐chained cytosolic acid soluble polyphosphate. The role of this polyphosphate fraction in detoxification of heavy metal ions is discussed.