Inorganic polyphosphate as an integral part of alkaline phosphatase preparations.

Alkaline phosphatase (from chicken intestinal sources) was shown to contain a considerable amount of polyanionic phosphorus which was released by basic digestion. The polyanionic phosphorus of alkaline phosphatase is not associated with protein or polyalcohols and does not exhibit a visible or ultraviolet absorption spectrum. Alkaline phosphatase and abiogenic inorganic polyphosphate were found to incorporate 32P-orthophosphate under similar experimental conditions. It has been previously reported that this enzyme will incorporate 32P-orthophosphate into its protein phosphoserine without the apparent concomitant utilization of an energy source. This reported phosphorylation was immediately reversible upon dilution of the phosphorylated enzyme with unlabelled orthophosphate, which indicates that the initial phosphorylation was an exchange reaction. These observations suggest that this polyanionic phosphorus from alkaline phosphatase may be inorganic polyphosphate.     

Isolation and characterization of polyphosphates from the yeast cell surface

When cells of Saccharomyces fragilis are subjected to osmotic shock, they release a limited amount of inorganic polyphosphate into the medium, which represents about 10% of the total cellular content. The osmotic shock procedure causes no substantial membrane damage, as judged from the unimpaired cell viability, limited K⁺ leakage and low percentage of stained cells. It is therefore suggested that this polyphosphate fraction is localized outside the plasma membrane. The released polyphosphate fraction differs from the remaining cellular polyphosphates in two respects: the mean chain length of the shock-sensitive fraction is significantly higher than that of the total cellular polyphosphates and its metabolic turnover rate, subsequent to pulsing with [³²P]orthophosphate is much lower compared to the rest of the cellular polyphosphate. Incubation of intact cells with the anion exchange resin Dowex AG 1-X4 results in the release of high molecular weight polyphosphates. These results suggest that the osmotic shock-sensitive polyphosphate fraction has specific characteristics in both its cellular localization and metabolism.     

Sodium transport and phosphorus metabolism in sodium-loaded yeast: simultaneous observation with sodium-23 and phosphorus-31 NMR spectroscopy in vivo

Simultaneous 23Na and 31P NMR spectra were obtained from a number of yeast suspensions. Prior to NMR spectroscopy, the yeast cells were Na-loaded: this replaced some of the intracellular K+ with Na+. These cells were also somewhat P-deficient in that they had no polyphosphate species visible in the 31P NMR spectrum. In the NMR experiments, the Na-loaded cells were suspended in media which contained inorganic phosphate, very low Na+, and a shift reagent for the Na+ NMR signal. The media differed as to whether dioxygen, glucose, or K+ was present individually or in combinations and as to whether the medium was buffered or not. The NMR spectra revealed that the cells always lost Na+ and gained phosphorus. However, the nature of the Na+ efflux time course and the P metabolism differed depending on the medium. The Na+ efflux usually proceeded linearly until the amount of Na+ extruded roughly equalled the amount of NH4+ and orthophosphate initially present in the medium (external phosphate was added as NH4H2PO4). Thus, we presume this first phase reflects a Na+ for NH4+ exchange. The Na+ efflux then entered a transition phase, either slowing, ceasing, or transiently reversing, before resuming at about the same value as that of the first phase. We presume that this last phase involves the simultaneous extrusion of intracellular anions as reported in the literature. The phosphorus metabolism was much more varied. In the absence of exogenous glucose, the P taken up accumulated first as intracellular inorganic phosphate; otherwise, it accumulated first in the "sugar phosphate" pool. In most cases, at least some of the P left the sugar phosphate pool and entered the polyphosphate reservoir in the vacuole. However, this never happened until the phase probably representing Na+ for NH4+ exchange was completed, and the P in the polyphosphate pool never remained there permanently but always eventually reverted back to the sugar phosphate pool. These changes are interpreted in terms of hierarchical energy demands on the cells under the different conditions. In particular, the energy for the Na+ for NH4+ exchange takes precedence over that required to produce and store polyphosphate. This conclusion is supported by the fact that when the cells are "forced" to exchange K+, as well as NH4+, for Na+ (by the addition of 5 times as much K+ to the NH4+-containing medium), polyphosphates are never significantly formed, and the initial linear Na+ efflux phase persists possibly 6 times as long.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitative analysis of formate in solutions containing phosphate

Standard colorimetric methods based on the initial reduction of formate to formaldehyde were found to yield erratic results when applied to the analysis of millimolar concentrations of formate in a microbial culture medium. The source of interference was identified as inorganic orthophosphate inhibition of the magnesium/hydrochloric acid reduction stage. Passivation of magnesium by millimolar concentrations of phosphate is known to occur at low pH and it is proposed that this phenomenon is responsible for the inhibition of the reduction process. The presence of orthophosphate in biological extracts is almost universal and would lead to acceptance of spuriously low values for formate concentration if the previously unreported inhibitory effect is not recognized. The colorimetric method of Barker and Somers in which formate is reacted directly with 2-thiobarbituric acid to form the chromophore was evaluated and proved to be entirely free from interference by orthophosphate and other medium components. This method although less sensitive than the formate reduction methods is therefore suggested as the method of choice for the determination of formate in biological or other solutions containing phosphate.     

The biosorption of cadmium and cobalt and iron ions by yeast <Emphasis Type="Italic">Cryptococcus humicola</Emphasis> at nitrogen starvation

Yeasts Cryptococcus humicola accumulated cadmium, cobalt, and iron (~?50, 17, and 4% of the content in the medium, respectively) from the medium containing glucose, phosphate, and 2 mmol/L of metal salts. The effects of metal absorption on the levels of orthophosphate (Pi) and inorganic polyphosphate (polyP) varied for the metals under study. The levels of Pi and polyP increased in the case of cadmium and cobalt, respectively. In the case of iron, no changes in the levels of Pi and polyP were observed. Multiple DAPI-stained polyP inclusions were observed in the cytoplasm of cadmium-containing cells. The intensity of DAPI staining of the cell wall especially increased in case of cobalt and iron accumulation.     

The Relation between the Synthesis of Inorganic Polyphosphate and Phosphate Uptake by Chlorella vulgaris

During a period of phosphate starvation, the phosphate contentof cells of Chlorella vulgaris which had been grown in phosphate-richsolution, decreased. The levels of most phosphate fractionsdeclined, especially those of inorganic polyphosphates, whichat first accounted for about 5 per cent of the total phosphateand virtually disappeared after 36 h starvation. On return toa phosphate medium, phosphate was taken up at a much fasterrate than before starvation, with a striking increase in acid-solublepolyphosphate. The stimulated phosphate uptake and polyphosphateincrease have been shown to be specific effects of phosphatestarvation, occurred only when excess phosphate was suppliedand required light or air for the provision of energy. Therewas relatively little change in the concentrations of otherphosphate fractions, including orthophosphate. Inorganic polyphosphatewas found to be synthesized solely from phosphate absorbed fromthe medium. It is argued that polyphosphate synthesis is a consequenceof the stimulation of phosphate uptake, induced by the starvationperiod.     

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.     

Polyphosphate synthesis in yeast

Polyphosphate synthesis was studied in phosphate-starved cells of Saccharomyces cerevisiae and Kluyveromyces marxianus. Incubation of these yeasts for a short time with phosphate and either glucose or ethanol resulted in the formation of polyphosphate with a short chain length. With increasing incubation times, polyphosphates with longer chain lengths were formed. Polyphosphates were synthesized faster during incubation with glucose than with ethanol. Antimycin did not affect the glucose-induced polyphosphate synthesis in either yeast. Using ethanol as an energy source, antimycin A treatment blocked both polyphosphate synthesis and accumulation of orthophosphate in the yeast S. cerevisiae. However, in K. marxianus, polyphosphate synthesis and orthophosphate accumulation proceeded normally in antimycin-treated cells, suggesting that endogenous reserves were used as energy source. This was confirmed in experiments, conducted in the absence of an exogenous energy source.     

Recovery of exocellular acid phosphatase activity on Saccharomyces mellis after treatment of the organism with reagents that affect the cell surface

Derepressed cells of Saccharomyces mellis were treated in one of several different ways to either elute or inactivate the exocellular enzyme, acid phosphatase. The enzyme was either (i) eluted from resting cells with 0.5 m KCl plus 0.1% beta-mercaptoethanol, (ii) eluted from exponential phase cells by growing the organism in derepressing media containing 0.5 m KCl, or (iii) inactivated on exponential phase cells by adding sufficient acid or base to growth media to destroy the enzyme but not enough to kill the cells. These treatments did not affect viability. Treated cells were transferred to fresh growth media or some other reaction mixture, and the kinetics of recovery of acid phosphatase activity was studied. In these reaction mixtures, enzyme was synthesized only by actively growing cells. Treated resting cells were indistinguishable from untreated, repressed resting cells in that the organism inoculated into complete growth medium remained in the lag phase for approximately 6 hr before both growth and enzyme synthesis began. Exponential phase derepressed cells treated by method (ii) or (iii) were transferred to fresh medium under conditions that allowed growth to continue. The cells immediately started to manufacture enzyme at a rate greater than normal until the steady-state level was reached, thus demonstrating a feedback control system. Exponential phase repressed cells were also transferred to fresh derepressing media under conditions which sustained growth. Though these cells began to grow immediately, there was a lag before acid phosphatase synthesis began followed by a lengthy inductive period. The length of the period of induction could be correlated with the polyphosphate content of the cells. As the supply of polyphosphate neared exhaustion, the rate of synthesis increased rapidly until it was greater than normal; this differential rate was sustained until the steady-state concentration was reached. When derepressed cells grow in a medium containing 0.5 m KCl, some acid phosphatase activity is found free in the culture fluid and some remains firmly attached to the cells despite the presence of the salt. The bound activity is subject to feedback control, but the steady-state level of this activity on the cells is only one-third that of the acid phosphatase on cells growing in nonsaline media. The extracellular phosphatase is produced at a rate that is several-fold greater than that of the exocellular enzyme in a nonsaline medium. The synthesis of the extracellular enzyme does not seem to be controlled by a feedback mechanism but is produced at a maximal rate as long as the cells are growing.     

Determination of the role of polyphosphate in transport-coupled phosphorylation in the yeast Saccharomyces cerevisiae

The role of polyphosphate in 2-deoxy-D-glucose transport was studied in yeast cells, pulse-labeled with [³²P]orthophosphate, by comparing the concentrations and specific activities of polyphosphate, orthophosphate and 2-dGlc-phosphate. When 2-dGlc transport was measured under aerobic conditions, it appeared that polyphosphate replenished the orthophosphate pool, indicating that polyphosphate has, at least mainly, an indirect role in sugar phosphorylation. Also in cells with a reduced respiratory capacity, due to a treatment with antimycin A, no direct role for polyphosphate in 2-dGlc transport could be detected. Under these conditions, only a very limited breakdown of polyphosphate occurred, probably because of the small decrease in the orthophosphate concentration.Abbreviations 2-dGlc 2-deoxy-D-glucose - P_i orthophosphate - P_n polyphosphate - SP sugar phosphate     

Volutin Granules in Zoogloea ramigera

Zoogloea ramigera, a gram-negative bacterium found in activated sludge, formed volutin granules when excess orthophosphate was added to a phosphate-starved culture. These volutin granules were stainable by hydrogen sulfide after lead acetate treatment and extractable by N-perchloric acid but were not adsorbed by activated charcoal. They appeared to consist of inorganic polyphosphate. Optimum granule formation in the arginine broth required 10 g of glucose, 3 mg of phosphate, and 1 to 20 mg of magnesium per liter of medium. At an Mg(2+) concentration of 1 mg/liter, very large granules appeared which often appeared to fill the cell. An excess of glucose, orthophosphate, or magnesium reduced granule formation. In the absence of sulfate, moderate granulation occurred in arginine broth before the addition of excess orthophosphate; granulation did not increase after the addition of phosphate.     

Phosphorylation of uridine with inorganic phosphates

Phosphorylation of uridine by heating with inorganic orthophosphates can take place through reaction of nucleoside with thermally produced polyphosphate or direct reaction of nucleoside with orthophosphate or a combination of both. The relative importance of the two pathways varies with the orthophosphate and temperature.     

31P NUCLEAR MAGNETIC RESONANCE STUDY OF INTRACELLULAR PHOSPHATE POOLS AND POLYPHOSPHATE METABOLISM IN HETEROSIGMA AKASHIWO (HADA) HADA (RAPHIDOPHYCEAE)1

The effects of starvation and subsequent addition of phosphate-containing medium on the phosphate metabolic intermediates were studied by ³¹P-NMR spectroscopy of perchloric acid extracts and intact cells of Heterosigma akashiwo (Hada) hada. When orthophosphate in the medium was completely depleted the medium was enriched with orthophosphate (4.5 μM). In the phosphate starved condition, the P cell quota was 76 fmol-cell⁻¹ and the major components of phosphate intermediates were phosphodiester, sugar phosphate and orthophosphate (P_i) After addition of P_i' rapid uptake of P_i was observed and the P cell quota increased to 108 fmol. cell⁻¹ in 2 h, 134 fmol. cell⁻¹ in 5 h and 222 fmol. cell⁻¹ in 1 day after addition of phosphate. The ³¹P-NMR spectrum indicated that a major portion of P was stored as polyphosphate, in which the average chain length of polyphosphate increased from 10 to 20 phosphate residues in one day after addition of P_i-     

RIBONUCLEIC ACID-POLYPHOSPHATE FROM ALGAE: III. HYDROLYSIS STUDIES

RNA-polyphosphate was isolated from synchronous Chlorella cells.After each of a series of hydrolytic treatments, RNA-polyphosphatewas chromatographically analyzed by means of a two-column ion-exchangesystem. Alkaline hydrolysates contained primarily ribonu-cleotides,pyrophosphate, and tripolyphosphate. Acid hydrolysates containedribonucleotides, purine bases, ribonucleosides, orthophosphate,and an unknown, inorganic, phosphorus-containing compound (X-P).Treatments with pancreatic ribonuclease, spleen phosphodiesterase,and yeast polyphosphatase left large amounts of RNA-polyphosphatefragments. Treatment with venom phosphodiesterase yielded ahigh molecular weight inorganic polyphosphate fraction freefrom RNA. Such material was hydrolyzed to pyro- and tripolyphosphateby potassium hydroxide, to orthophosphate and an unknown compoundX-P by perchloric acid, and to ortho-, pyro-, and tripolyphosphateby hydroxylamine under ester hydrolysis conditions. Syntheticinorganic polyphosphate was stable to potassium hydroxide andhydroxylamine under the same conditions and yielded only orthophosphateupon perchloric acid hydrolysis. Both natural and syntheticpolyphosphates were hydrolyzed to low molecular weight fragmentsby yeast polyphosphatase. The evidence at present indicatesthat in Chlorella polyphosphate is not a simple phosphate anhydridechain. (Received June 14, 1965; )     

Betaine Transport Imparts Osmotolerance on a Strain of Lactobacillus acidophilus

Unlike most Lactobacillus acidophilus strains, a specific strain, L. acidophilus IFO 3532, was found to grow in rich medium containing 1 M sodium acetate, KCl, or NaCl. This strain could also grow with up to 1.8 M NaCl or 3 M nonelectrolytes (fructose, xylose, or sorbitol) added. Thus, this strain was tolerant to osmotic pressures up to 2.8 osM. A search for an intracellular solute which conferred osmoprotection led to the identification of glycine betaine (betaine). Betaine was accumulated to high concentrations in cells growing in MRS medium supplemented with 1 M KCl or NaCl. Uptake of [¹⁴C]betaine by L. acidophilus 3532 cells suspended in buffer was stimulated by increasing the medium osmotic pressure with 1 M KCl or NaCl. The accumulated betaine was not metabolized further; transport was relatively specific for betaine and was dependent on an energy source. Other lactobacilli, more osmosensitive than strain 3532, including L. acidophilus strain E4356, L. bulgaricus 8144, and L. delbrueckii 9649, showed lower betaine transport rates in response to an osmotic challenge than L. acidophilus 3532. Experiments with chloramphenicol-treated L. acidophilus 3532 cells indicated that the transport system was not induced but appeared to be activated by an increase in osmotic pressure.     

Elution of Acid Phosphatase from the Cell Surface of Saccharomyces mellis by Potassium Chloride.

Weimberg, Ralph (Northern Regional Research Laboratory, Peoria, Ill.), and William L. Orton. Elution of acid phosphatase from the cell surface of Saccharomyces mellis by potassium chloride. J. Bacteriol. 90:82-94. 1965.-Acid phosphatase of Saccharomyces mellis may be eluted from intact resting cells by 0.5 m KCl or other salts. However, the enzyme is not eluted at higher salt concentrations of about 2 m unless a thiol, such as beta-mercaptoethanol, is included in the reaction mixture. These treatments do not significantly affect viability of the cells. Neutral compounds like sorbitol or sucrose cannot substitute for ionic compounds in eluting the enzyme from resting cells. Furthermore, the neutral compounds are also inadequate for stabilizing the protoplast structure. It is suggested that the enzyme is held on the cell surface by a combination of electrostatic forces and disulfide bonds. Thiol alone dissociates protein and carbohydrate from the cell surface, but the eluate has no acid phosphatase activity. Salts also remove protein and carbohydrate from the cell surface, but the amount of protein removed is considerably less than that dissociated by thiol. A concentration of 0.5 m KCl elutes more protein than does a 2 m concentration, and enzymatic activity is present only in the 0.5 m KCl eluate. The carbohydrate eluted by either reagent has been identified as a mannan. Conditions for eluting acid phosphatase from acetonedried cells of S. mellis are essentially the same as those for resting cells. Significantly, though, thiol is required at all salt concentrations to dissociate the enzyme. Pretreatment of the cells with thiol, followed by KCl, elutes acid phosphatase, whereas the reverse procedure does not. Acid phosphatase is excreted by growing cells of S. mellis into growth media if the medium contains 0.25 m KCl. The total yield of enzymatic activity may be 8 to 10 times greater than is usually present on derepressed cells grown in a salt-free medium. The enzyme can be precipitated from the culture fluid with acetone. The acetone-precipitated fraction contains mannan and protein in a ratio of 12:1 by weight. Partial purification of the enzyme by calcium phosphate gel and elution resulted in an enzyme fraction in which the specific activity on the basis of protein increased 12-fold, and the carbohydrate-protein ratio was reduced to 1:1.     

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."     

31P NUCLEAR MAGNETIC RESONANCE STUDY OF INTRACELLULAR PHOSPHATE POOLS AND POLYPHOSPHATE METABOLISM IN HETEROSIGMA AKASHIWO (HADA) HADA (RAPHIDOPHYCEAE)1

The effects of starvation and subsequent addition of phosphate-containing medium on the phosphate metabolic intermediates were studied by ³¹P-NMR spectroscope of perchloric acid extracts and intact cells of Heterosigma akashiwo (Hada) Hada. When orthophosphate in the medium was completely depleted the medium was enriched with orthophosphate (4.5 μM). In the phosphate starved condition, the P cell quota was 76 fmol·cell⁻¹ and the major components of phosphate intermediates were phosphodiester, sugar phosphate and orthophosphate (P_i). After addition of P_i, rapid uptake of P_i was observed and the P cell quota increased to 108 fmol·cell⁻¹ in 2 h, 134 fmol·cell⁻¹ in 5 h and 222 fmol·cell⁻¹ in 1 day after addition of phosphate. The ³¹P-NMR spectrum indicated that a major portion of P was stored as polyphosphate, in which the average chain length of polyphosphate increased from 10 to 20 phosphate residues in one day after addition of P_i.     

Novel method for enzymatic synthesis of CMP-NeuAc

A novel method for synthesizing CMP-NeuAc was established. We first confirmed that the putative neuA gene of Haemophilus influenzae, identified by its whole genome sequence project, indeed encodes CMP-NeuAc synthetase (EC 2.7.7.43). The enzyme requires CTP as a cytidylyl donor for cytidylylation of NeuAc. The enzyme was coupled with an enzymatic CTP-generating system from CMP and inorganic polyphosphate as a sole phospho-donor driven by the combination of polyphosphate kinase and CMP kinase, where phosphorylation of CMP is done by the combined activity expressed by both enzymes, and subsequent phosphorylation of CDP by polyphosphate kinase itself occurred efficiently. When CMP-NeuAc synthetase of H. influenzae, polyphosphate kinase, and CMP kinase were added to the reaction mixture containing equimolar concentrations (15 mM) of CMP and NeuAc, and polyphosphate (150 mM in terms of phosphate), CMP-NeuAc was synthesized up to 10 mM in 67% yield.     

Influence of ions and tonicity of RNA metabolism in Tetrahymena pyriformis

Net RNA degradation occurs in Tetrahymem pyrifmmis when this ciliate is suspended in a non-nutrient medium. The quantity and quality of the excretion products is at least partially under the control of the ionic content and the tonicity of the cellular environment. The excretion of ultraviolet-absorbing materials was found to be elevated by sodium ions in a medium isotonic to the culture fluid, or by a hypertonic environment. Magnesium counteracted these effects. In isotonic suspension, sodium and magnesium ions lowered orthophosphate excretion; however, sodium altered the nature of the phosphate products so that acidlabile phosphates were also excreted rather than solely orthophosphate. Similar results were obtained in a hypertonic environment with or without sodium. The degree of purine and pyrimidine loss from the cells in all conditions of suspension was reflected in the amount of RNA degraded. The ion and tonicity effects apparently reflect events which alter the stability of the RNA and the properties of the membrane system, resulting in changes in both the rate of RNA degradation and the nature of the excreted products. The rates of orthophosphate excretion appear to be affected by changes in the acid-base balance within the cell which may be governed by the cation levels. The manipulation of the ionic content and tonicity of the medium offers a convenient method for obtaining cells reduced in RNA content.