Intracellular Phosphate Levels Research Articles

Possible role for carbamyl phosphate in the control of liver glycogen synthesis

Isotopic data suggested a possible correlation between the activation of liver glycogen synthesis and some unstable intermediate. Carbamyl phosphate was one candidate considered. L-Norvaline, an inhibitor of ornithine transcarbamylase, is known to increase intracellular carbamyl phosphate levels. L-Norvaline augmented the increase in glycogen synthesis caused by L-glutamine and L-alanine, and also induced considerable glycogen synthesis with ammonia as the nitrogen source.

Arterial metabolism as studiedin vitroby NMR: Preliminary results in normotensive and hypertensive aortas

Arterial tissue has been analysed by 31P-, 13C-, 23Na- and 1H-NMR spectroscopy. Rabbit thoracic aortas were mounted on a system with perfusate circulation and studied in basal conditions. Phosphorus spectra remained stable for hours and showed low levels of phosphocreatine (PCr) compared to skeletal, cardiac or even to nonvascular smooth muscle. Significant levels of sugar-phosphates (SP), phosphodiesters (PDE) were detected, as well as occasionnally a peak in the diphosphodiester region. Experiments with phosphate-free perfusate demonstrated a very low level of intracellular inorganic phosphate. As expected from previous data, free ADP levels in tonic arterial tissue were found much higher than in any other muscle. Addition of norepinephrine into the perfusate induced transient decrease in ATP and PCr levels, associated with an increased production of phosphorylated intermediates. At the early stage of renovascular hypertension, aortic energetic pattern was characterized by an increased ADP/ATP ratio. Natural abundant 13C spectra were recorded from dog aortic fragments and showed mainly resonances attributed to fatty components. After addition of a shift-reagent, dysprosium tripolyphosphate, 23Na-NMR allowed separation of intra- and extracellular Na of perfused rabbits aortas. Proton NMR of lyophilized aortic fragments revealed several peaks originating from biologically relevant molecules, lactate, creatine, taurine... These preliminary data demonstrate the feasability of multinuclear NMR spectroscopy of vascular tissue and are suggestive of the potential of the method when it will be combined with monitoring of functional parameters.

Phosphate regulation of phosphatases in submerged cultures of Claviceps purpurea 129 producing clavine alkaloids

Claviceps purpurea strain 129 was cultivated under submerged conditions in a sucrose-citrate medium containing high (36.8 mM) or low (1.84 mM) KH2PO4 concentrations. The permeabilized cells and culture supernatants contained alkaline and acid phosphatases. In the medium containing a high phosphate concentration, the synthesis of extracellular phosphatases was repressed, but that of cellular phosphatases was not. Extracellular phosphatases, especially alkaline phosphatases, were derepressed by transferring the mycelium into a phosphate-free medium. This derepression was inhibited by cycloheximide. In the presence of cycloheximide, the activities of the cellular phosphatases decreased markedly, indicating turnover of these enzymes. The cellular acid phosphatase was inhibited by phosphate (0.025 M–0.1 M) and NaF (0.01 M) while the cellular alkaline phosphatase was only inhibited by phosphate. Both cellular and extracellular alkaline phosphatases were more sensitive to repression by phosphate than the acid phosphatases. The alkaloid synthesizing enzymes were: a) present in mycelia grown in high levels of phosphate and b) activated by decreasing the intracellular phosphate level.

Influence of accumulated phosphate on culture growth and formation of cinnamoyl putrescines in medium-induced cell suspension cultures of Nicotiana tabacum

The inorganic phosphate of the liquid nutrient medium was completely taken up by freshly inoculated cells of Nicotiana tabacum L. within the first 2 d of culture. Thus intracellular ortho-phosphate concentrations of approx. 0.06 M were accumulated, which upon growth of the cultures were diluted by cell division and subsequent cell growth. Cells from different stages of the growth cycle containing progressively decreasing levels of phosphate were transferred to a phosphate-free medium which normally stimulates the formation of cinnamoyl putrescines. The resulting accumulation of these compounds was inversely correlated with the intracellular phosphate level, whereas a direct linear relationship in the phosphate concentration was found with further growth in the phosphate-free medium.

Carotid body O2 chemoreception and mitochondrial oxidative phosphorylation.

The effect on carotid chemoreceptor afferents of oligomycin, an inhibitor of mitochondrial oxidative phosphorylation that does not affect energy conservation, was studied in 20 cats that were anesthetized, paralyzed, and artificially ventilated. Responses of single or a few chemoreceptor afferents to changes in arterial O2 tension (PaO2) at constant arterial CO2 tension were recorded. In addition, responses to nicotine, cyanide, and antimycin A or carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone (FCCP) were tested in normoxia. Oligomycin (50-500 microgram) was administered by close intra-arterial injection, and the same tests were repeated at timed intervals. Initially, oligomycin caused vigorous stimulation of carotid chemoreceptor activity. Subsequently, although the afferent fibers were still active and could be vigorously stimulated by nicotine, they no longer responded to changes in PaO2 or to doses of cyanide, antimycin A, or FCCP. These results separate stimulation of chemoreceptor afferents by hypoxia and metabolic inhibitors and uncouplers from that by nicotine and suggest that intact oxidative phosphorylation, required for maintenance of the intracellular high-energy phosphate levels, forms the basis of O2 chemoreception in the carotid body.

Relationship between Energy-dependent Phosphate Uptake and the Electrical Membrane Potential in Lemna gibba G1

High rates of phosphate uptake into phosphate-starved Lemna gibba L. G1 were correlated with a high membrane potential (pd = -220 millivolts). In plants maintaining a low pd (-110 millivolts), the uptake rate was only 20% of that of high-pd plants. At the onset of phosphate transport, the membrane of high-pd plants was transiently depolarized. This effect was much smaller in low-pd plants. Light stimulated phosphate uptake and the repolarization upon phosphate-induced depolarization, especially in plants grown without sucrose. The phosphate uptake rate was optimal at pH 6 and decreased with increasing pH, corresponding to the phosphate-induced pd changes. Phosphate starvation stimulated the uptake and increased the phosphate-induced depolarization, thus indicating that phosphate uptake depends on the intracellular phosphate level. It is suggested that uptake of monovalent phosphate in Lemna gibba proceeds by an H(+) cotransport dependent on the proton electrochemical potential difference and, hence, on the activity of an H(+) -extrusion pump.

The role of phosphate in the action of vitamin D on the intestine.

The response of chick intestine to vitamin D and its metabolites was studied in an organ culture preparation of chick ileum explants. Both 25-hydroxycholecalciferol (25-OHD(3)) at a concentration of 20 ng/ml or greater and 1,25-dihydroxycholecalciferol [1,25-(OH)(2)D(3)] at a concentration of 50 pg/ml or greater stimulated the rate of accumulation of [(32)P]phosphate and (45)Ca by the explants and the incorporation of [(3)H]thymidine into DNA. The accumulation of [(32)P]phosphate by the explants was against a concentration gradient and inhibited by ouabain and dinitrophenol. Two saturable mechanisms appeared to mediate the cellular accumulation of phosphate with K(a) of 0.0047 and 0.125 mM, respectively. The V(max) of the lower affinity transport mechanism was accelerated by 1,25-(OH)(2)D(3). Actinomycin D (5.0 mug/ml) did not block the intestinal response to 1,25-(OH)(2)D(3) stimulation of both [(32)p]phosphate and (45)Ca accumulation. Significant stimulation of [(32)P]phosphate accumulation was observed 30 min after the addition of 1,25-(OH)(2)D(3), preceding the sterol-induced increase in the rate of (45)Ca uptake by 30 min and the sterol-induced increase in [(3)H]thymidine incorporation into DNA by 150 min. Increasing extracellular phosphate concentration to 3.0 mM increased [(3)H]thymidine incorporation into DNA and the rate of (45)Ca uptake by the explants. Reducing extracellular phosphate concentration to 0.05 mM attenuated the response of the explants to 1,25-(OH)(2)D(3). From these observations it is postulated that the primary action of vitamin D sterols in the intestine is to enhance the ability of the mucosal cell to accumulate phosphate. The data suggest that restoration of intracellular phosphate levels may then permit expression of the cells' response to vitamin D sterols.

Colchicine inhibition of plasma protein release from rat hepatocytes.

Colchicine, both in vitro and in vivo, inhibits secretion of albumin and other plasma proteins. In vitro, secretion by rat liver slices is inhibited at 10-minus 6 M with maximal effect at 10-minus 5 M. Inhibition of secretion is accompanied by a concomitant retention of nonsecreted proteins within the slices. Colchicine does not inhibit protein synthesis at these concentrations. Vinblastine also inhibits plasma protein secretion but lumicolchicine, griseofulvin, and cytochalasin B do not. Colchicine also acts in vivo at 10-25 mumol/100 g body weight. Inhibition of secretion is not due to changes in the intracellular nucleotide phosphate levels. Colchicine, administered intravenously, acts within 2 min and its inhibitory effect lasts for at least 3 h. Colchicine has no effect on transport of secretory proteins in the rough or smooth endoplasmic reticulum but it causes these proteins to accumulate in Golgi-derived secretory vesicles.

Purification and Characterization of a Polyphosphate Kinase from Arthrobacter atrocyaneus

Polyphosphate kinase, an enzyme which incorporated the gamma-phosphate of ATP into long-chain polyphosphate molecules, was purified more than 700-fold from Arthrobacter atrocyaneus by ammonium sulphate fractionation, DEAE-cellulose column chromatography and Ssphadex G-200 gel filtration. The enzyme had a broad pH optimum at 6-0 to 7-0 and required Mn2+ or Mg2+, histone, and inorganic phosphate for activity. The Km for Mn-ATP was 0-53 mM, and for inorganic phosphate was 1-67 mM. Free ATP concentrations greater than 8 muM inhibited the enzyme. Free Mn2+ or Mg2+ concentrations greater than 2 mM or 6 mM, respectively, were also inhibitory. Activity was strongly inhibited by 4 mM-ADP, 1 mM-PP1 or 20 mM-NaF. The effect of ADP might have resulted from reversing the equilibrium of the kinase reaction. The activation by phosphate ions might indicate a role for the enzyme in regulating intracellular phosphate levels or maintaining a phosphorus reserve. The level of enzymic activity in the bacteria responded to changes in inorganic phosphate concentration in the medium. Basic proteins, such as protamine, could substitute for histone as activator. Proteins such as casein or bovine serum albunim would also substitute for histone but only in the absence of inorganic phosphate. The presence of a protein might be necessary to form a complex with the product, thus preventing reversal of the reaction in vitro. The reaction product was characterized, and found to be labile in hydroxylamine, base, and acid at 100 degrees C. It behaved as a long-chain-polyphosphate molecule on chromatography in an Ebel's solvent. The enzymic activity was therefore not that of a protein kinase.

Additional biochemical studies on erythrocytes containing hemoglobin Sabine

Additional studies have been carried out on erythrocytes containing an unstable hemoglobin (Sabine). Increased levels of hexokinase and glyceraldehyde phosphate dehydrogenase, together with a high intracellular inorganic phosphate level and a high ADP ATP ratio, account for the very rapid glycolytic rate in these cells. The high intracellular inorganic phosphate level appears to be a consequence of increased uptake of phosphate into the cells, with uptake coupled to activities of two membrane enzymes, glyceraldehyde phosphate dehydrogenase and phosphoglycerate kinase. In vitro incubation studies over a period of 14 hours indicate that these cells maintain intracellular K + and Na + levels satisfactorily. These studies do not, however, indicate whether rates of influx or efflux of these cations are modified in these abnormal erythrocytes.

Glycolysis and respiration in Ehrlich ascites tumour cells. IV. Pasteur effect, malonate Pasteur effect and Crabtree effect.

Summary.1. The aerobic inhibition of glucose utilization i. e. the Pasteur effect in Ehrlich ascites tumour cells decreased when the concentration of inorganic phosphate in the medium and the level of intracellular inorganic phosphate increased. It was accompanied by a rise in acid soluble phosphate esters. A higher aerobic than anaerobic glucose uptake was observed in IAA inhibited cells, or by incubation in a strong phosphate buffer (negative Pasteur effect).2. In ascites tumour cells, partially blocked with malonate, fumarate in contrast to citrate, stimulated oxygen uptake and inhibited glycolysis (the malonate Pasteur effect). This effect also decreased when the concentration of inorganic phosphate in the medium and the level of intracellular, inorganic phosphate increased. It was inhibited by DNP and anaerobiosis.3. In glycolyzing cells, blocked with malonate, fumarate further enhanced the acid labile phosphate and ATP, in spite of the accompanying rise in oxygen uptake. Thus the Crabtree effect could not be explained by lack of total cellular inorganic phosphate or phosphate acceptors.4. The Pasteur and Crabtree effects in Ehrlich ascites tumour cells are explained by the preferential channeling of limited a‐mounts of cellular inorganic phosphate which is to some extent carried by adenine nucleotides into certain reactions at the expense of others.

Dependence on Light of the Reversible Uptake of Inorganic Phosphate by Hydrodictyon

IN the colony-forming green alga, Hydrodictyon reticulatum, uptake and metabolism of phosphate have been followed up by ordinary analytical methods (fractionation by the conventional trichloracetic acid technique, determination of phosphates with a modified procedure of Fiske and Subbarow1). The predominant feature in the changes of the phosphate fractions during a long-term culture with intermittent illumination (12 hr. light: 12 hr. darkness) is a marked increase in the large cells of the alga of the inorganic phosphate-level during the time of illumination. A comparable light-dependent accumulation of ions has been reported for other objects (reviewed by Brauner2), most recently for chloride by van Lookeren-Campagne3. In the case of Hydrodictyon, however, the accumulation of inorganic phosphorus in the light is reversible4, since the low level of intra-cellular phosphate is completely restored during the dark time. This is shown in Fig. 1. The changes of the inorganic phosphorus have been found in the cells and, at the same time, in the surrounding nutrient solution. The differences in the phosphorus-level are not directly induced by pH. changes in the medium.