Phosphate dependency of phosphofructokinase 2

Experiments performed at micromolar concentrations of inorganic phosphate support the conclusion that liver phosphofructokinase 2 would be completely inactive in the absence of inorganic phosphate or arsenate. The concentration of inorganic phosphate that allowed half-maximal activity decreased with increasing pH, being approximately 0.11 mM at pH 6.5 and 0.05 mM at pH 8. The effect of phosphate was to increase V and to decrease Km for fructose 6-phosphate, without affecting Km for ATP. Citrate and P-enolpyruvate inhibited the enzyme non-competitively with fructose 6-phosphate and independently of the concentration of inorganic phosphate. Phosphorylation of the enzyme by the catalytic subunit of cyclic-AMP-dependent protein kinase did not markedly modify the phosphate requirement and its effect of inactivating phosphofructokinase 2 could not be counteracted by excess phosphate. A nearly complete phosphate dependency was also observed with phosphofructokinase 2 purified from Saccharomyces cerevisiae or from spinach leaves. By contrast, the fructose 2,6-bisphosphatase activity of the liver bifunctional enzyme was not dependent on the presence of inorganic phosphate. Phosphate increased this activity about threefold when measured in the absence of added fructose 6-phosphate and a half-maximal effect was reached at approximately 0.5 mM phosphate. Like glycerol phosphate, phosphate counteracted the inhibition of fructose 2,6-bisphosphatase by fructose 6-phosphate, but a much higher concentration of phosphate than of glycerol phosphate was required to reach this effect.     

Subcellular localization of enzymes inStreptomyces aureofaciens and its alteration by benzyl thiocyanate

Mycelia of a low- and a high production strain ofStreptomyces aureofaciens were converted into protoplasts and divided into five subcellular fractions in order to localize exopolyphosphatases (EC 3.6.1.11), triphosphatase (EC 3.6.1.25), inorganic diphosphatase (EC 3.6.1.1), apyrase (EC 3.6.1.5) and glucokinase (EC 2.7.1.2). The highest specific activity of enzymes hydrolyzing polyphosphates was found in cytoplasmic vesicles and membranes. Triphosphatase was detected in the periplasmic fraction. Periplasmic vesicles and cytoplasm exhibited a high activity of diphosphatase. Apyrase was found mainly in the fractions of membranes and cytoplasmic vesicles. Glucokinase was a cytoplasmic enzyme. The enzymes were released from membrane structures into cytoplasm or periplasmic space if benzyl thiocyanate (10 μm) was present in the growth medium.     

Kinetics of yeast growth and enzyme syntheses in a phosphate-limited continuous culture

Summary The effect of a deficiency of inorganic phosphate on the growth rate and on the invertase and phosphatase activities inSaccharomyces carlsbergensis was studied in a chemostat culture using a synthetic medium in which ethanol was the sole carbon source.The kinetic relationship between the growth rate and both the rates of phosphate uptake and the ethanol consumption agreed well with the threshold model but not the multicative model. The invertase activity of the yeast increased as the dilution rate decreased. As the phosphate concentration in the feed was reduced, the enzyme synthesis increased remarkably. Acid phosphatase activity was repressed completely above a critical molecular ratio, 0.015, of monopotassium phosphate to ethanol in the feed medium. As the phosphate concentration in the feed decreased, the maximum specific enzyme activity increased and the corresponding optimum dilution rate decreased. These experimental changes in enzyme synthesis were expressed mathematically using the modified operon models for enzyme regulation in terms of two fractions of limited inorganic phosphate; one which affects growth and the other which is incorporated in excess by the cells.Nomenclature A ethanol concentration in the culture (mM) - a, b, c, d exponents in the operon model - D dilution rate (h^–1) - E enzyme concentration in the culture (enzyme unit l^–1) - K_a, K_b, K_c, K_d, k equilibrium constants used in the operon model, see Toda (1976b) - o operator gene - P inorganic phosphate concentration in the culture (mM) - Pi limited inorganic phosphate concentration in the cells (mmole inorganic phosphate/g dry weight of cell) - Q specific enzyme activity, no units: (E/X)/(E/X)_max - Q_c, Q_d as defined in Eq. 12 - R repressor - r regulator gene - X cell concentration in the culture (dry cell weight l^–1) Greek Letters molecular ratio of inorganic phosphate to ethanol in the feed medium (mole/mole) - specific growth rate (h^–1) - A specific uptake rate of ethanol (mmole/g cell·h) - P specific uptake rate of inorganic phosphate (mmole/g cell·h) Suffix crit critical value - f feed - max maximum - min minimum - t total - 1, 2 number of species Superfix eff effective for cell growth - exc excess - str structural     

Adaptation of the cells ofEscherichia coli to the presence of hydroxyurea increases the level of inorganic pyrophosphatase activity

Hydroxyurea, an inhibitor of ribonucleoside diphosphate reductase, completely arrested the net synthesis of DNA for 3–4 h, when it was added in 30 mM concentration to growing cultures ofEscherichia coli K12. Thereafter the net synthesis of DNA started again, although slowly, and simultaneously with it the formation of inorganic pyrophosphatase activity was stimulated leading to a 2-fold increase in the specific activity of the enzyme in 2–3 h. Subsequently cell division began again. In this way a new steady state, stable in the presence of hydroxyurea, was reached. This new state was characterized by the high specific activity of inorganic pyrophosphatase, a small but constant amount of DNA/cell mass (1/4 of the normal value), and large elongated cells. All these changes were slowly reversed during 5–6 h, when the cells were transferred into a drug-free medium.The activity of isoleucyl-tRNA synthetase, assayed as a control, did not change significantly in the presence of hydroxyurea.Hydroxyurea had no effect on the activity of inorganic pyrophosphatase in vitro.     

Relationship between the carbohydrate metabolism ofStreptomyces aureofaciens and the biosynthesis of chlortetracycline

The effect of interrupted aeration on the biosynthesis of chlortetracycline (CTC) was investigated. The culture is most sensitive to interruption in aeration when between the 6th and 12th hour of growth. Then even short interruptions will result in a pronounced suppression of CTC biosynthesis. Using glucose labelled at carbon 1 and at carbon 6 with¹⁴C it could be demonstrated that the interruption in aeration brings about a decrease in the activity of the pentose shunt during breakdown of sugar in the course of subsequent cultivation. A similar effect can be induced by increasing the level of inorganic phosphate in the medium. It was shown by studying the interaction of benzyl thiocyanate and interruption of aeration on the biosynthesis of CTC that benzyl thiocyanate antagonizes the unfavourable effect of interrupted aeration. Its presence will prevent a drop in CTC production by a culture aerated with interruptions. The relationship between the enzymatic reactions of the pentose shunt and the mechanism of chlortetracycline biosynthesis is discussed.     

Regulation of cephalosporin synthesis in Cephalosporium acremonium by phosphate and glucose

The regulation of cephalosporin synthesis in Cephalosporium acremonium was studied in a simple chemically-defined medium with glucose as the carbon source. Antibiotic synthesis depended on the phosphate content of the medium. At phosphate concentrations above 2.75 mM maximum antibiotic titres were not reached while glucose uptake and growth rates were increased. Phosphate exerted its effect indirectly by regulating the rate of glucose consumption. The negative effect of high phosphate concentrations could be overcome completely by controlling the sugar supply in fed-batch and chemostat experiments. High actual concentrations of phosphate or of glucose alone had no direct negative effect on antibiotic synthesis.     

Effect of Inorganic Phosphate on the Extracellular Acid Phosphatase Activity of Tobacco Cells Cultured in vitro

Acid phosphatase activity in culture medium of tobacco cells growing in suspension increased with the age of the culture from which the medium was obtained. The increase in the activity was accelerated by omitting inorganic phosphate from nutrient medium, and it was depressed by addition of inorganic phosphate or cycloheximide. Amylase and β-galactosidase activities were not induced by the omission of inorganic phosphate. It was concluded that derepression of acid phosphatase synthesis was involved in the increase in the extracellular acid phosphatase activity upon inorganic phosphate depletion.     

Dependence of the maximum temperature for growth of Saccharomyces cerevisiae on nutrient concentration

Anacystis nidulans (Synechococcus) was maintained in a medium of low phosphate concentration (0.1 mM) and grew with a normal doubling time of 5 hrs at 30°C. Such cultures ahd a normal pigment composition and alkaline phosphatase was detectable at low specific activities only.The onset of phosphate-limited growth occurred when the phosphate concentration in the medium fell to a value below 4 M (the limit of accurate determination by the assay method used) and resulted in increases in alkaline phosphatase activity, reaching a final 10 to 15 fold increase in specific activity after a period of several hours. Marked changes in the overall pigment composition occurred in this period of growth restriction. The addition of phosphate to such cultures resulted in a halt in synthesis of the enzyme and the restoration of normal pigmentation before growth resumed at the normal rate.Several organic phosphate esters could replace inorganic phosphate for growth and were also hydrolyzed by the partially purified enzyme, but growth rates were characteristically lower and the specific activity only 3 to 4 fold higher than in cultures grown in phosphate excess.Studies with the partially purified enzyme suggested that it differed in some of its properties from other alkaline phosphatases described in the literature.Abbreviations Used pNP pnitrophenol - pNPP pnitrophenylphosphate     

INTERACTIONS AMONG PHOSPHATE UPTAKE,PHOTOSYNTHESIS, AND CHLOROPHYLL FLUORESCENCE IN NUTRIENT‐LIMITED CULTURES OF THE CHLOROPHYTE MICROALGA DUNALIELLA TERTIOLECTA1

Phosphate‐limited and phosphate‐sufficient continuous cultures of the marine chlorophyte microalga Dunaliella tertiolecta Butcher were examined for their responses to the addition of phosphate. Phosphate‐limited cultures showed a marked quenching of chl fluorescence following a pulse of phosphate. This response was absent from cells growing under phosphate‐sufficient conditions. Both the extent of fluorescence quenching (where present) and the initial rate of change in quenching were dependent on the concentration of phosphate added to cell suspensions and on the degree of limitation (growth rate in continuous culture). The addition of phosphate also brought about a transient decrease in photosynthetic oxygen evolution and a stimulation in respiration, which were relaxed as the added phosphate was depleted from the external medium. The applicability of using nutrient‐induced fluorescence transients as a tool to identify the nutrient status of phytoplankton populations is discussed.     

Control of teichoicand teichuronic acid biosynthesis in Bacillus subitlis 168trp−: Evidence for repression of enzyme synthesis and inhibition of enzyme activity

Phosphate starvatiion induced teichuronic acid synthesis in cells of Bacillus subtilis 168trp^? which had previously been grown with excess phophate. This induction was prevented when protein synthesis was inhibited immediately prior to phosphate starvation and under these conditions cells continued to form teichoic acid. The converse was true when phosphate was added to cells previously grown in phosphate-limited chemostat. The increase in teichoic acid synthesis normally following phosphate addition was prevented by chlorampehnicol or amino acid starvation and cells continued to make teichuronic acid. The suggestion that repression of enzyme synthesis is involved in controlling the type of wall polymer made was supported by the low levels of UDP-glucose dehydrogenase found in cells grown with excess phosphate and of CDP-glycerol pyrophosphorylase in phophate-limited cells. The greater amounts of teichoic acid made under phosphate limitation and of teichuronic acid with excess phosphate when protein synthesis was also inhibited indicated that modulation of enzyme activity occurs. Glycerol starvation of a glycerol-requiring mutant did not derepress teichuronic acid synthesis, indicating that glycerol-containing intermediates do not act as repressors.     

Microbial Phospholipid Synthesis as a Marker for Microbial Protein Synthesis in the Rumen

Phosphate uptake into intracellular inorganic phosphorus and cellular phospholipids and the relationship between cell growth and phospholipid synthesis were studied with suspensions of washed ruminal bacteria in vitro with (33)P-phosphorus. It was shown that ruminal bacteria accumulated inorganic phosphate at a low rate when incubated without substrate. Upon the addition of substrate, the rate of inorganic phosphorus uptake into the cells increased markedly, and phospholipid synthesis and cell growth commenced. There was a highly significant relationship (r = 0.98; P < 0.01) between phospholipid synthesis and cell growth. The specific activity of the intracellular inorganic phosphorus did not equilibrate with phosphorus medium. When ruminal contents from sheep fed a high or low protein diet were incubated in vitro, the rate of (33)P incorporation into microbial phospholipids was higher for the high protein diet. Since there was a high relationship between phospholipid synthesis and growth, rumen contents were collected before and various times after feeding and incubated with (33)P-phosphorus in vitro. The short-term, zero time approach was used to measure the rate of microbial phospholipid synthesis in whole rumen contents. In these studies the average specific activity of the intracellular inorganic phosphorus was used to represent the precursor pool specific activity. Microbial phospholipid synthesis was then related to protein (N x 6.25) synthesis with appropriate nitrogen-to-phospholipid phosphorus ratios. Daily true protein synthesis in a 4-liter rumen was 185 g. This represents a rate of 22 g of protein synthesized per 100 g of organic matter digested. These data were also corrected for ruminal turnover. On this basis the rate of true protein synthesis in a 4-liter rumen was 16.1 g of protein per 100 g of organic matter digested. This value represents a 30-g digestible protein-to-Mcal digestible energy ratio which is adequate for growing calves and lambs.     

Biosynthesis of ethylene: the effect of phosphate*

Abstract Biosynthesis of ethylene in tomato and avocado fruit slices, carrot root, pea seedling and tomato shoot segments, Penicillium expansum and Escherichia coli was found to be inhibited by inorganic phosphate. Compared with microbial systems, relatively high concentrations of phosphate in the incubating medium were necessary to bring about a significant inhibition of ethylene production in higher plants. The degree of inhibition in higher plants correlated with the increased internal cellular concentration of phosphate and not with that of the incubating medium. Phosphate concentrations inhibitory for ethylene biosynthesis did not affect the respiration of tomato fruit slices. The phosphate effect was reversible, confined to only the biological systems and was not due to a change in the ionic strength. The differential inhibitory effects of aminoethoxyvinylglycine on ethylene biosynthesis in tomato fruit slices of various stages of ripening, were markedly influenced by high phosphate concentrations. The data indicate a biological significance to the phosphate control of ethylene biosynthesis.     

The effect of phytase on the availability of P from myo-inositol hexaphosphate (phytate) for maize roots

The effect of adding phytase to the root medium of maize plants on the P-availability of added myo-inositol hexaphosphate (phytin) has been studied in pot experiments. When 40 mM phytin-P in nutrient solution was incubated in quartz-sand for 15 days in the absence of plants, 80% of it could be recovered from the solution as soluble organic P. Maize plants growing on this mixture assimilated P from phytin at rates comparable to those from inorganic phosphate (Pi). At a lower addition rate (2 mM phytin-P) only 10% was recovered in the soil solution, and plant growth was severely limited by P. At this low phytin level, the addition of phytase (10 enzyme units per kg sand) increased the plants' dry weight yield by 32%. The relative increases of the Pi concentration in the solution and of the amount of P in the plants were even higher, indicating that the observed growth stimulation was due to an increased rate of phytin hydrolysis. The enzyme-induced growth stimulation was also observed with plants growing in pots filled with soil low in P, when phytin was added. However, on three different soils the addition rates of phytin and phytase necessary for obtaining a significant phytase effect were both about 10 times higher than those required in quartzsand. It is concluded that the P-availability from organic sources can be limited by the rate of their hydrolytic cleavage.Abbreviation Pi inorganic phosphate     

Some aspects of xylose isomerase constitutive biosynthesis in <Emphasis Type="Italic">Arthrobacter nicotianae</Emphasis>

The specific features of biosynthesis of the cell-bound xylose isomerase by the actinobacterium Arthrobacter nicotianae BIM V-5 were studied. It was demonstrated that the constitutive synthesis of this enzyme in the studied bacteria, not subject to catabolite repression, was inhibited by xylulose, an intermediate product of xylose utilization and the final product of its enzymatic isomerization. Short-term experiments demonstrated that xylulose at a concentration of 0.005% almost completely repressed the xylose isomerase synthesis in A. nicotianae. This effect was independent of the time moment when the repressor was added to the cultivation medium and was not associated with its influence on the catalytic activity of the enzyme.     

Changes in the adenine nucleotide content of beef-heart mitochondrial F1 ATPase during ATP synthesis in dimethyl sulfoxide.

Beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me2SO. We have analyzed the adenine nucleotide content of the F1 ATPase during the time-course of ATP synthesis, in the absence of added medium nucleotide, and in the absence and presence of 10 mM inorganic phosphate. The enzyme used in these investigations was either pretreated or not pretreated with ATP to produce F1 with a defined nucleotide content and catalytic or noncatalytic nucleotide-binding site occupancy. We show that the mechanism of ATP synthesis in Me2SO involves (i) an initial rapid loss of bound nucleotide(s), this process being strongly influenced by inorganic phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and inorganic phosphate.     

Inhibition of Inorganic Pyrophosphatase from Escherichia coli with Inorganic Phosphate

The interaction of inorganic pyrophosphatase from E. coli with inorganic phosphate (P _i) was studied in a wide concentration range of phosphate. The apoenzyme gives two inactive compounds with P _i, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate. The phosphorylation occurs when P _i is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex. The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium. The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.     

Factors influencing the activity of cellular alkaline phosphatase during growth and sporulation ofBacillus cereus

Alkaline phosphatase (EC 3.1.3.1) is synthesized in media with a low phosphate concentration (0.37 mmm of total and 19 μm of inorganic phosphate, respectively) already during the exponential phase of growth ofBacillus cereus. The enzyme is repressed by higher phosphate concentrations (3.7 mm) during the whole growth period; during sporogenesis the enzyme activity in cells slightly increases even under these conditions. During growth the enzyme is not secreted into the medium, a minor amount being released after cessation of growth. The enzyme activity can be increased by adding Zn²⁺ ions (10 μm). When during growth without phosphate the pH of the medium decreases below 5.0, the enzyme activity temporarily decreases and growth is slowed down, followed by a subsequent increase of the enzyme activity. In this case the onset of sporulation is also delayed.     

Phosphatase from Proteus mirabilis

Cell-free preparations of Proteus mirabiliscontained a phosphatase (EC 3.1.3.1) whose activity surpassed that of alkaline phosphatase from Escherichia coli. Phosphatase was also found in the culture liquid of P. mirabilis. The composition of proteins displaying enzyme activity was assayed by polyacrylamide gel electrophoresis. Enzyme synthesis was studied at various stages of bacterial growth. Biosynthesis of phosphatase in P. mirabilis(similarly to that found in other bacteria) was shown to be induced under conditions of inorganic phosphate deficiency in the medium.     

Effect of phosphate concentration on the production of dextransucrase by<Emphasis Type="Italic"> Leuconostoc mesenteroides</Emphasis> NRRL B512F

Leuconostoc mesenteroides NRRL B512F is the main strain used in industrial fermentations to produce dextransucrase and dextran. This process has been studied since the Second World War, when it was used as blood plasma expander. A study about the effect of phosphate concentration on cell propagation in a semicontinuous shake-flask culture is described in this work. Dextransucrase is obtained by fermentation of the Leuconostoc mesenteroides NRRL B512F in the presence of sucrose as substrate, a nitrogen source (corn liquor or yeast extract) and minerals. Phosphate is currently used in order to buffer the culture medium. Cell propagation can be done through a repeated batch culture, where dilution in a fresh medium is made with relatively short periods. The standard medium for dextransucrase production is prepared using 0.1 M of K₂HPO₄. In this work the level of phosphate was increased to 0.3 M, and an increase on biomass and on the enzyme activity was found when phosphate enriched medium was used. Higher phosphate buffer concentration was also able to keep the pH values above 5.0 during the entire process, avoiding enzyme denaturation.