Magnesium-Dependent stimulation of protein synthesis by the insulin mimic,pervanadate

The insulin mimic, peroxide of vanadate (pervanadate), stimulated ³⁵S-methionine incorporation into Xenopus oocyte protein in a Mg²⁺-dependent manner. Reducing the extracellular Mg²⁺ concentration from 1.0 to 0.1 mM decreased the pervanadate-stimulated component of incorporation by 35%; with 0.01 mM Mg²⁺ or lower, the pervanadate-stimulated component was abolished. In addition, reducing extracellular Mg²⁺ to 0.01 mM inhibited about 50% of the insulinstimulated component of methionine incorporation. Mg²⁺ depletion had no effects on incorporation in controls or when protein synthesis was stimulated by Zn²⁺ or bovine growth hormone. Thus, not all substances that stimulated protein synthesis showed a dependence on extracellular Mg²⁺. Reducing extracellular Ca²⁺ had no effects on methionine incorporation in control cells or in cells stimulated by pervanadate or insulin. When oocytes maintained in a paraffin oil medium were brought into contact with a 0.5 m?I droplet of buffer containing the Mg²⁺ indicator dye, mag-fura-2, and pervanadate, apparent droplet Mg²⁺ decreased rapidly, indicating net uptake by the cells. Insulin also caused a net uptake of Mg²⁺. In contrast, apparent extracellular Mg²⁺ was constant when cells were in contact with droplets containing no effectors. Together, these data indicate that extracellular Mg²⁺, but not Ca²⁺, is involved in the stimulation of protein synthesis by pervanadate, and to a lesser extent by insulin. Pervanadate appears to induce a net uptake of Mg²⁺, and this change in membrane transport may be an early event in signalling the increase in translation. © 1995 Wiley-Liss, Inc.     

Active hexose uptake in Lemna gibba G1

Growth of autotrophically growing duck-weeds (Lemna gibba L., G1) was stimulated by sucrose. The rate of respiration increased when plants had been grown on sucrose (8.7 mol O₂ g^-1 fresh weight (FW) h^-1) and was reduced after growth without sucrose in the dark or under longday conditions (2.5 mol O₂ g^-1 FW h^-1). Photosynthesis was induced already by low light intensities (0.1 klx).Short-time application of glucose or sucrose stimulated respiration in proportion to the hexose uptake rate. Sucrose is probably not taken up as the disaccharide. The transported sugar species after addition of sucrose are its hexose moieties produced by the high activity of the cell wall invertase. Fructose stimulated to a lesser extent; mannitol induced no enhancement; 2-deoxyglucose slightly inhibited O₂ uptake. After mild carbon starvation of the plants the uptake of glucose and 3-O-methylglucose proceeded without any lag phase, with similar saturation kinetics in both cases. The initial uptake rate at substrate saturation was 2.6 mol glucose g^-1 FW h^-1 in the dark. Light stimulated hexose uptake by 2 to 3 times. The results show that Lemna gibba has an energy-dependent constitutive system for hexose uptake.Abbreviation FW fresh weight - LD long day - SD short day     

The effects of α- and β-adrenergic agents,Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent V_max without altering the K_m. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl₂ also increased uptake. Lowering of the perfusate Ca²⁺ concentration from 1.27 to 0.1 mM Ca²⁺, addition of the Ca²⁺ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca²⁺ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca²⁺, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca²⁺ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca²⁺ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca²⁺ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca²⁺ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca²⁺-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca²⁺-dependent and a Ca²⁺-independent component. Reversal studies suggest an additional role for Ca²⁺ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Insulin stimulation of glucose transport and metabolism in a human Wilms' tumor-derived myoblast-like cell Line: Modulation of hormone effects by glucose deprivation

The effects of insulin and glucose on parameters of metabolism were investigated in myoblast-like (MBL) cells, a human myoblast-like cell line derived from a Wilms' tumor. Insulin responses were studied after 4 hr pre-incubation in serum free media, with or without 5 mM glucose. Insulin was added during the last 2 hr. Glucose starvation markedly increased basal glucose transport (measured as 2-deoxyglucose uptake) as well as the net uptake of [¹⁴C]glucose and [¹⁴C]glucose incorporation into glycogen. Insulin stimulated net glucose uptake and incorporation into glycogen in a dose-dependent manner in glucose-fed and starved cells. These insulin responses were markedly enhanced in glucose-starved cells. Insulin accelerated 2-deoxyglucose transport in glucose-fed cells but did not further stimulate basal glucose transport in glucose-deprived cells. Insulin increased the incorporation of [³H]leucine into protein in glucose-fed or -starved MBL cells equally. The dose of insulin required for half-maximal insulin responses was similar for all parameters studied. Cycloheximide did not prevent the increased basal glucose incorporation in glucose-starved cells, but markedly inhibited the insulin response, while in glucose-fed cells, cycloheximide stimulated basal glucose incorporation. We conclude that MBL cells resemble fibroblasts in their insulin-independent stimulation of glucose transport in response to glucose-deprivation; when provided with glucose, they respond to insulin like fibroblasts. However, after brief glucose-starvation, the stimulated glucose transport system is no longer insulin-responsive in MBL cells, while pathways leading to the synthesis of macromolecules demonstrate preserved or enhanced stimulation by insulin, suggesting that these cells may serve as models to study the regulation of receptor-response coupling by the metabolic milieu.     

Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl^– uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations50 mM. In contrast, in the absence of Cl^–, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid, 200 M Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl^–/vanadate ratios may result from competition between vanadate and Cl^– for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - FC fusicoccin - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid We thank Drs. R.T. Leonard (University of California, Riverside, USA) and K.A, Rubinson (Yellow Springs, Oh., USA) for helpful comments on the research, Janet Sherwood (Harvard University) for excellent plant care, and Angela Ciamarra, Anne Gershenson, Gustavo Lara (Harvard University) and Orit Tal (Hebrew University) for valuable technical assistance. This research was supported by a grant from the National Science Foundation (DCB-8904041) to S.M.A.     

Phosphate absorption rates and adenosine 5'-triphosphate concentrations in corn root tissue

The correlations between ATP concentration in corn (Zea mays) root tissue and the rate of phosphate absorption by the tissue have been examined. Experimental variation was secured with 2,4-dinitrophenol, oligomycin, mersalyl, l-ethionine, 2-deoxyglucose, N₂ gassing and inhibition of protein synthesis. It is concluded that ATP could be the energy source for potassium phosphate absorption, but only if the transport mechanism possesses certain properties: oligomycin-sensitivity; creation of a proton gradient susceptible to collapse by uncouplers; phosphate transport via a mersalyl-sensitive Pi⁻-OH⁻ transporter; good activity at energy charge as low as 0.4; short enzymatic half-life for the ATPase or phosphate transporter; a linked mechanism for K⁺-H⁺ exchange transport, possibly electrogenic.     

Dependence of the kinetics of secondary active transports in yeast on H+-ATPase acidification

Acidification of the external medium of the yeast Saccharomyces cerevisiae, mainly caused by proton extrusion by plasma membrane H⁺-ATPase, was inhibited to different degrees by D₂O, diethylstilbestrol, suloctidil, vanadate, erythrosin B, cupric sulfate and dicyclohexylcarbodiimide. The same pattern of inhibition was found with the uptake of amino acids, adenine, uracil, and phosphate and sulfate anions. An increase of the acidification rate by dioctanoylglycerol also increased the rates of uptake of adenine and of glutamic acid. In contrast, a decrease of the membrane potential at pH 4.5 from a mean of -40 to -20 mV caused by 20 mm KC1 had no effect on the transport rates. The ATPase-deficient mutant S. cerevisiae pmal-105 showed a markedly lower uptake of all the above solutes as compared with the wild type, while its membrane potential and pH were unchanged.Other types of acidification (spontaneous upon suspension; K⁺ stimulated) did not affect the secondary uptake systems.     

The mitogen-activated protein kinase pathway contributes to vanadate toxicity in vascular smooth muscle cells

Vanadate has been considered in the treatment of diabetes because of its insulin-like effects. However, it has severe toxic effects in both animal and man. In cultured cells, vanadate can either cause death or be growth stimulatory, depending on the cell type and growth conditions. Here, we report that in baboon aortic smooth muscle cells (SMCs), vanadate induced p42/p44 mitogen-activated protein kinase (MAPK) activity. This effect was abolished in the presence of the specific MAPK kinase (MAPKK) inhibitor PD098059. Although activation of p42/p44^MAPK/MAPKK is generally thought to be necessary for proliferation, in SMCs, vanadate did not promote DNA synthesis and inhibited thymidine incorporation stimulated by platelet-derived growth factor (PDGF)-BB in a dose dependent fashion (IC₅₀: 30 M). Prolonged exposure to vanadate exerted cytotoxic effects. Cells retracted, rounded up and detached from the substratum. These vanadate-induced morphological changes were blocked in the presence of PD098059. The addition of PDGF-BB further activated p42/p44^MAPK/MAPKK in the presence of vanadate and substantially increased vanadate toxicity. We conclude from these observations that activation of the p42/p44^MAPK/MAPKK signalling module contributes to the cytotoxic effects induced by vanadate.     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs: I. Chlorfluazuron,diflubenzuron, and teflubenzuron inhibit incorporation but not uptake of [14C]N-acetyl-D-glucosamine

The purpose of this study was to determine the effects of potent inhibitors of chitin synthesis on an organ culture test system as a basis for determining the mode of action of such compounds. Consequently, we investigated the action of chlorfluazuron (CFA), diflubenzuron (DFB), and teflubenzuron (TFB) on uptake and incorporation into chitin of [¹⁴C]N-acetyl-D-glucosamine ([¹⁴C]GlcNAc) in wing imaginal discs cultured in vitro. Spodoptera frugiperda wing imaginal discs provided a highly responsive test system for studying the inhibition of ecdysteroid-dependent chitin synthesis in a target tissue in vitro. All three inhibitors blocked ecdysteroid-dependent [¹⁴C]GlcNAc incorporation into chitin by the wing imaginal discs. The effectiveness of the inhibitors was not affected by the time of their application, i.e., exposures before, during, or after 20-hydroxyecdysone treatment were equally effective in inhibiting chitin synthesis. Thus, exposure of freshly dissected discs to CFA for periods as short as 15 min inhibited approximately 90% of the chitin synthesis measured 72 h later. In contrast to previous in vivo studies all three inhibitors were similar in their effectiveness in vitro. However, while all three compounds inhibited [¹⁴C]GlcNAc incorporation in a similar dose-dependent manner, only DFB and TFB reduced but did not block uptake of GlcNAc. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Independent control of selected insulin-sensitive cell membrane and intracellular functions — the linkage of cell membrane and intracellular events controlled by insulin

Summary The effects of pH, oxidation reduction compounds and trypsin on insulin binding, hexose transport, and activation of glycogen synthase were studied utilizing rat adipocytes. In this paper the effect of pH is examined; while in the subsequent two papers the effects of glutathione and trypsin are examined. Increase in pH from 6 to 8.5 increased labelled glucose oxidation, 2-deoxyglucose transport as well as labelled insulin binding to the receptor. Enhanced insulin binding was due to an increased rate of association k₊₁ with no effect the rate of dissociation k₋₁ resulting in a decreased equilibrium dissociation constant K_D. Glycogen synthase activity was unaffected by increase in pH when adipocytes were incubated with or without glucose. Insulin in contrast to pH was effective in increasing the activity of glycogen synthase. With 2-deoxyglucose, % glycogen synthaseI activity was increased by an increase in pH. Glycogen synthase activity was thus stimulated by insulin by the direct mechanism, previously termed mechanism 1, involving the formation of a chemical mediator, and clearly distinguishable from the activation of hexose transport, previously termed mechanism 2(1). Increase in labelled glucose oxidation and in 2-deoxyglucose transport with increased pH, as well as insulin stimulation, was abolished by preincubation with trypsin, or cytochalasin B; suggesting that trypsin-sensitive and cytochalasin B-binding protein(s) presumably in the plasma membrane are involved in these effects of pH. Since increase in pH alone activates cell membrane-mediated hexose transport and insulin receptor binding under conditions where glycogen synthase is not activated, increase in pH acts presumably by a non-mediator mechanism. Insulin acts at the membrane to enhance further the effects of increased pH, via a mediator mechanism.     

Stimulation of acid invertase activity by indol-3yl-acetic acid in tissues undergoing cell expansion

The soluble acid invertase activity of young, excised P. vulgaris internodal segments fell when they were incubated in water, and their elongation ceased within 6–7 h. IAA (10 M) promoted segment elongation and stimulated an increase in the specific activity of acid invertase to a level greater than that originally present. The rate of segment elongation in the presence of IAA was closely and positively correlated with the specific activity of the enzyme. Optimum concentration of IAA for both elongation and stimulation of invertase activity was 10 M. Concurrent protein synthesis was necessary for these responses to IAA. Segments cut from mature, fully-elongated internodes did not responsd to IAA.Inclusion of Ca²⁺, vanadate or mannitol in the incubation medium abolished IAA-induced segment elongation but did not inhibit the stimulation of acid invertase activity by IAA. Auxin-induced elongation and acid invertase activity were both substantially increased in the presence of up to 25 mM D-glucose or up to 50 mM sucrose. Inclusion of either sugar in the medium considerably increased tissue hexose concentrations. Under some circumstances cell growth and invertase synthesis may compete for available hexose substrate.It is concluded that IAA-induced promotion of acid invertase in P. vulgaris internodal segments is not simply an indirect consequence of removal of end-product (hexose) during IAA-induced cell growth and that a more direct action of IAA on enzyme turnover is involved.     

Multiple effects of mercuric chloride on hexose transport in Xenopus oocytes.

HgCl(2) had both stimulatory and inhibitory effects on [(3)H]2-deoxyglucose (DG) uptake in Xenopus laevis oocytes. The Hg dose response was complex, with 0.1-10 microM Hg increasing total DG uptake, 30-50 microM Hg inhibiting, and concentrations >100 microM increasing uptake. Analyses of the effects of Hg on DG transport kinetics and cell membrane permeability indicated that low concentrations of Hg stimulated mediated uptake, intermediate concentrations inhibited mediated uptake, but high Hg concentrations increased non-mediated uptake. 10 microM Hg increased the apparent V(max) for DG uptake, but caused little or no change in apparent K(m). Phenylarsine oxide prevented the increase in DG uptake by 10 microM Hg, suggesting that the increase was due to transporter recruitment. Microinjecting low doses of HgCl(2) into the cell increased mediated DG uptake. Higher intracellular doses of Hg increased both mediated and non-mediated DG uptake. Both insulin and Hg cause cell swelling in isotonic media and, for insulin, this swelling has been linked to the mechanism of hormone action. Osmotically swelling Xenopus oocytes stimulated DG transport 2-5-fold and this increase was due to an increased apparent V(max). Exposing cells to 10 microM Hg or 140 nM insulin both increased cellular water content by 18% and increased hexose transport 2-4-fold. These data indicate that low concentrations of Hg and insulin affect hexose transport in a similar manner and that for both an increase cellular water content could be an early event in signaling the increase in hexose transport.     

Announcement

Phosphate concentration was found to control the biosynthesis of the antibiotic candicidin by resting cells of Streptomyces griseus. Phosphate concentrations above 1 mM decreased the rate of incorporation of [¹⁴C]propionate and [¹⁴C]p-aminobenzoic acid into candicidin in relation to the concentration of phosphate. The inhibitory effect of phosphate on incorporation of labeled precursors into candicidin was not caused by inhibition of cellular uptake of precursors. Protein synthesis, sensitive to chloramphenicol, was not affected by phosphate levels that inhibit antibiotic synthesis. Similarly, phosphate concentrations inhibitory to antibiotic synthesis did not affect rifampinsensitive RNA synthesis.     

Vanadate binding to the (Na + K)-ATPase

A particulate (Na + K)-ATPase preparation from dog kidney bound [⁴⁸V]-ortho-vanadate rapidly at 37°C through a divalent cation-dependent process. In the presence of 3 mM MgCl₂ theK _d was 96 nM; substituting MnCl₂ decreased theK _d to 12 nM but the maximal binding remained the same, 2.8 nmol per mg protein, consistent with 1 mol vanadate per functional enzyme complex. Adding KCl in the presence of MgCl₂ increased binding, with aK _0.5 for KCl near 0.5 mM; the increased binding was associated with a drop inK _d for vanadate to 11 nM but with no change in maximal binding. Adding NaCl in the presence of MgCl₂ decreased binding markedly, with anI ₅₀ for NaCl of 7 mM. However, in the presence of MnCl₂ neither KCl nor NaCl affected vanadate binding appreciably. Both the nonhydrolyzable, ,-imido analog of ATP and nitrophenyl phosphate, a substrate for the K-phosphatase reaction that this enzyme also catalyzes, decreased vanadate binding at concentrations consistent with their acting at the low-affinity substrate site of the enzyme; the presence of KCl increased the concentration of each required to decrease vanadate binding. Oligomycin decreased vanadate binding in the presence of MgCl₂, whereas dimethyl sulfoxide and ouabain increased it. With inside-out membrane vesicles from red blood cells vanadate inhibited both the K-phosphatase and (Na + K)-ATPase reactions; however, with the K-phosphatase reaction extravesicular K⁺ (corresponding to intracellular K⁺) both stimulated catalysis and augmented vanadate inhibition, whereas with the (Na + K)-ATPase reaction intravesicular K⁺ (corresponding to extracellular K⁺) both stimulated catalysis and augmented vanadate binding.     

Glucose uptake in human and animal muscle cells in culture

Human muscle cells were grown in culture from satellite cells present in muscle biopsies and fusion-competent clones were identified. Hexose uptake was studied in fused myotubes of human muscle cells in culture and compared with hexose uptake in myotubes of the rat L6 and mouse C2C12 muscle cell lines. Uptake of 2-deoxyglucose was saturable and showed an apparent Km of about 1.5 mM in myotubes of all three cell types. The Vmax of uptake was about 6000 pmol/(min.mg protein) in human cells, 4000 pmol/(min.mg protein) in mouse C2C12 muscle cells, and 500 pmol/(min.mg protein) in L6 cells. Hexose uptake was inhibited approximately 90% by cytochalasin B in human, rat, and mouse muscle cell cultures. Insulin stimulated 2-deoxyglucose uptake in all three cultures. The hormone also stimulated transport of 3-O-methylglucose. The sensitivity to insulin was higher in human and C2C12 mouse myotubes (half-maximal stimulation observed at 3.5 X 10(-9) M) than in rat L6 myotubes (half-maximal stimulation observed at 2.5 X 10(-8) M). However, insulin (10(-6) M) stimulated hexose uptake to a larger extent (2.37-fold) in L6 than in either human (1.58-fold) or mouse (1.39-fold) myotubes. It is concluded that human muscle cells grown in culture display carrier-mediated glucose uptake, with qualitatively similar characteristics to those of other muscle cells, and that insulin stimulates hexose uptake in human cells. These cultures will be instrumental in the study of human insulin resistance and in investigations on the mechanism of action of antidiabetic drugs.     

ATP-dependent carbon transport in perfused Chara cells

Carbon transport across the plasma membrane, and carbon fixation were measured in perfused Chara internodal cells. These parameters were measured in external media of pH 5·5 and pH 8·5, where CO₂ and HCO₃^- are, respectively, the predominant carbon species in both light and dark conditions. Cells perfused with medium containing ATP could utilize both CO₂ and HCO₃^- from the external medium in the light. Photosynthetic carbon fixation activity was always higher at pH 5·5 than at pH 8·5. When cells were perfused either with medium containing hexokinase and 2-deoxyglucose to deplete ATP from the cytosol (HK medium) or with medium containing vanadate, a specific inhibitor of the plasma membrane H⁺_-ATPase (V medium), photosynthetic carbon fixation was strongly inhibited at both pH 5·5 and 8·5. Perfusion of cells with medium containing pyruvate kinase and phosphoenolpyruvate (PEP) to maximally activate the H⁺_-ATPase (PK medium), stimulated the photosynthetic carbon fixation activities. Oxygen evolution of isolated chloroplasts and the carbon fixation of cells supplied ¹⁴C intracellularly were not inhibited by perfusion media containing either hexokinase and 2-deoxyglucose or vanadate. The results indicate that Chara cells possess CO₂ and HCO₃^- transport systems energized by ATP and sensitive to vanadate in the light. In the dark, intact cells also fix carbon. By contrast, in cells perfused with medium containing ATP, no carbon fixation was detected in 1 mol m ^-3 total dissolved inorganic carbon (TDIC) at pH 8·5. By increasing TDIC to 10 mol m^-3, dark fixation became detectable, although it was still lower than that of intact cells at 1mol m^-3 TDIC. Addition of PEP or PEP and PEP carboxylase to the perfusion media significantly increased the dark-carbon fixation. Perfusion with vanadate had no effect on the dark-carbon fixation.     

Upregulation of the Na+-Coupled Phosphate Cotransporters NaPi-IIa and NaPi-IIb by B-RAF

B-RAF, a serine/threonine protein kinase, contributes to signaling of insulin-like growth factor IGF1. Effects of IGF1 include stimulation of proximal renal tubular phosphate transport, accomplished in large part by Na⁺-coupled phosphate cotransporter NaPi-IIa. The related Na⁺-coupled phosphate cotransporter NaPi-IIb accomplishes phosphate transport in intestine and tumor cells. The present study explored whether B-RAF influences protein abundance and/or activity of type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb. cRNA encoding wild-type NaPi-IIa and wild-type NaPi-IIb was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type B-RAF, and electrogenic phosphate transport determined by dual-electrode voltage clamp. NaPi-IIa protein abundance in Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified by chemiluminescence. Moreover, in HEK293 cells, the effect of B-RAF inhibitor PLX-4720 on NaPi-IIa cell surface protein abundance was quantified utilizing biotinylation of cell surface proteins and western blotting. In NaPi-IIa-expressing Xenopus oocytes, but not in oocytes injected with water, addition of phosphate to extracellular bath generated a current (I _P), which was significantly increased following coexpression of B-RAF. According to kinetic analysis, coexpression of B-RAF enhanced the maximal I_P. Coexpression of B-RAF further enhanced NaPi-IIa protein abundance in the Xenopus oocyte cell membrane. Treatment of HEK293 cells for 24 h with PLX-4720 significantly decreased NaPi-IIa cell membrane protein abundance. Coexpression of B-RAF, further significantly increased I_P in NaPi-IIb-expressing Xenopus oocytes. Again, B-RAF coexpression enhanced the maximal I_P. In conclusion, B-RAF is a powerful stimulator of the renal and intestinal type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb, respectively.     

PHOSPHATE UPTAKE BY SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYCEAE): ENHANCEMENT BY CALCIUM ION1

The influence of metallic, cations (added at 10 μM-1 mM) on the uptake of orthophosphate from 0.2–10 μM solution by Synechococcus leopoliensis (Racib.) Komarek was investigated. All cations tested except Mg²⁺ and Zn²⁺ stimulated phosphate uptake. The most pronounced stimulation of phosphate uptake was caused by Ca²⁺·Ca²⁺ markedly decreased the half-saturation concentration for orthophosphate uptake, apparently by acting upon the metabolic processes of phosphate transport into the cell. Phosphate did not influence Ca²⁺ fluxes across the cell-surface.     

Metabolic regulation in the senescing tobacco leaf: I. Changes in pattern of p incorporation into leaf disc metabolites

Changes in phosphate metabolism were explored in discs from tobacco (Nicotiana tabacum) leaves of three contrasting types: green leaves which were fully expanded and attached to the plant, leaves which had yellowed following excision and dark starvation, and leaves which had yellowed while attached to the plant. 2,4-Dinitrophenol at 10⁻⁵m stimulated the respiration rate of discs from green and yellow-detached leaves only slightly, but markedly stimulated that of discs from yellow-attached leaves. Following a 10-minute uptake period the incorporation of ³²P-orthophosphate into phosphate esters and lipids of discs from yellow-detached leaves was resistant to 2,4-dinitrophenol, whereas in discs from green and yellow-attached leaves it was inhibited by 2,4-dinitrophenol. Incorporation into a salt-soluble fraction containing unidentified nucleotide material showed converse behavior in that it was stimulated by 2,4-dinitrophenol in discs from green and yellow-attached leaves; in discs from yellow-detached leaves it was resistant to 2,4-dinitrophenol. In discs from yellow-detached and yellow-attached leaves there was a shift in the labeling pattern of phosphate esters toward increased label in hexose phosphates at the expense of adenine nucleotides, 3-phosphoglycerate, and phosphoenolpyruvate. It is concluded that incorporation into phosphate esters in discs from yellow-detached leaves is by substrate level phosphorylation coupled to enhanced aerobic glycolysis. In discs from yellow-attached leaves, on the other hand, incorporation depends on oxidation phosphorylation, and it is suggested that the shift in labeling pattern is caused by senescence-induced changes in activity of glycolytic enzymes.