A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway

Vitamin B₆ is essential for cellular functions and growth due to its involvement in important metabolic reactions. Humans and other mammals cannot synthesize vitamin B₆ and thus must obtain this micronutrient from exogenous sources via intestinal absorption. The intestine, therefore, plays a central role in maintaining and regulating normal vitamin B₆ homeostasis. Due to the water-soluble nature of vitamin B₆ and the demonstration that transport of other water-soluble vitamins in intestinal epithelial cells involves specialized carrier-mediated mechanisms, we hypothesized that transport of vitamin B₆ in these cells is also carrier mediated in nature. To test this hypothesis, we examined pyridoxine transport in a model system for human enterocytes, the human-derived intestinal epithelial Caco-2 cells. The results showed pyridoxine uptake to be 1) linear with time for up to 10 min of incubation and to occur with minimal metabolic alteration in the transported substrate, 2) temperature and energy dependent but Na⁺ independent, 3) pH dependent with higher uptake at acidic compared with alkaline pHs, 4) saturable as a function of concentration (at buffer pH 5.5 but not 7.4) with an apparent Michaelis-Menten constant (K_m) of 11.99 ± 1.41 µM and a maximal velocity (V_max) of 67.63 ± 3.87 pmol · mg protein^-1 · 3 min^-1, 5) inhibited by pyridoxine structural analogs (at buffer pH 5.5 but not 7.4) but not by unrelated compounds, and 6) inhibited in a competitive manner by amiloride with an apparent inhibitor constant (K_i) of 0.39 mM. We also examined the possible regulation of pyridoxine uptake by specific intracellular regulatory pathways. The results showed that whereas modulators of PKC, Ca⁺²/calmodulin (CaM), and nitric oxide (NO)-mediated pathways had no effect on pyridoxine uptake, modulators of PKA-mediated pathway were found to cause significant reduction in pyridoxine uptake. This reduction was mediated via a significant inhibition in the V_max, but not the apparent K_m, of the pyridoxine uptake process. These results demonstrate, for the first time, the involvement of a specialized carrier-mediated mechanism for pyridoxine uptake by intestinal epithelial cells. This system is pH dependent and amiloride sensitive and appears to be under the regulation of an intracellular PKA-mediated pathway. vitamin B₆; intestinal transport; transport regulation; Caco-2 cell     

Effects of Light on Transport by Azolla pinnata

Effects of light flux density (LFD) during growth and uptakeassay on induction of transport system and kinetics of transport were studied using the Azolla pinnata-Anabaena azollae association (Azolla). Theinduction and uptake kinetics of the transport system were determined using an automated system that measuredthe NO₃ concentration in the growth medium as a function oftime, using an on-line high performance liquid chromatograph(HPLC) with a UV-VIS detector. Full induction of the transport system required about 1.5 to 2.0 h and occurred without any apparent lag phase,regardless of the LFD provided. The level of induction of transport of Azolla grown at 600 µmol m^–2s^–1 LFD was higher than for that grown at 100 µmolm^–2 s^–1. Similarly, 600 µmol m^–1 s^–1LFD during the assay resulted in a higher level of inductionthan did 100 umol m^–2 s^–1. An increase in the LFDeither during the growth or the assay period increased the uptake rate; however, an increase in LFD duringthe latter period had greater effect. Azolla grown and assayedat 600 umol m^–2 s^–1 had the highest uptake rate. The uptake rate at 50 cm³ m^–3ambient CO₂ concentration was initially higher than at 305 cm³m^–3, but the uptake rate decreased rapidly with time andeventually dropped below that at 305 cm³ m^–3 CO₂. Thesedata suggest that the energy required for transport in Azolla may bypass the photosynthetic CO₂ fixationand carbon-cycling. Key words: carbon dioxide, concentration dependence, light flux density, uptake     

Nitrogen-13 Studies of Nitrate Fluxes in Barley Roots: II. EFFECT OF PLANT N-STATUS ON THE KINETIC PARAMETERS OF NITRATE INFLUX

Influx of nitrate into the roots of intact barley plants wasfollowed over periods of 1–15 min using nitrogen-13 asa tracer. Based on measurements taken over 15 min from a rangeof external nitrate concentrations (0·2–250 mmolm^–3), the kinetic parameters of influx, I_max and K_m, werecalculated. Compared with plants grown in the presence of nitrate throughout,plants that had been starved of N for 3 d showed a significantlygreater value ofI_max for ¹³N-nitrate influx (by a factor of1·4–1·8), but a similar value of K_m (12–14mmol m^–3). Pre-treating N-starved plants with nitratefor about 5 h further increased the subsequent rate of ¹³N-nitrateinflux, but had little effect in the unstarved controls. Allowingfor this induction of additional nitrate transport, the differencein rates of nitrate influx in control and N-starved plants wassufficient to account for the previously-observed differencein net uptake by the two groups of plants. In barley plants grown without any exposure to nitrate, butwith ammonium as N-source, both I_max and K_m for subsequent ¹³N-nitrateinflux were significantly decreased (by about one-half) comparedwith the corresponding nitrate-grown controls. The importance of changes in the rate of influx in the regulationof net uptake of nitrate is discussed. Key words: Ion transport, nitrate, influx, kinetic parameters, N-deficiency     

The Transport and Metabolism of Urea in Chara australis: I. PASSIVE DIFFUSION, SPECIFIC TRANSPORT AND METABOLISM OF UREA, THIOUREA AND METHYLUREA

Most of the urea entering Chara australis cells is rapidly metabolizedto produce CO₂, which diffuses out of the cells into the surroundingmedium. A simple and convenient apparatus to measure both the¹⁴C-urea retained by cells and the ¹⁴CO₂ released into the mediumwas developed and used in a study of urea transport in Chara.The permeability coefficient for urea in the Chara plasmalemmawas estimated from the slope of an uptake versus concentrationfunction as 85 nm s^-1. Computer modelling of urea uptake andmetabolism suggests that this could be a 20% underestimate ofthe true value.The corresponding permeability coefficients forthiourea and N-methyl-urea were estimated in the same way as34 and 35 nm s^-1, respectively. These permeabilities are muchgreater than expected on the basis of either/water partitioncoefficients for the solutes and are consistent with the diffusionof urea and its similarly-sized analogues through aqueous poresin the plasmalemma.At external concentrations of urea less than20 mmol m^-3, the bulk of the uptake is effected by a specifictransport mechanism with an apparent K_m for urea of less than1.0 mmol m^-3. This transport system operates most rapidly withexternal pH in the range 6.5–7.5 and is influenced bythe nitrogen status of the cell.Evidence is produced here suggestingthat the specific transport of urea may be an active process. Key words: Chara, urea uptake, metabolism, diffusion, specific transport     

Potassium-Ammonium Uptake Interactions in Tobacco Seedlings

Short-term (< 12 h) uptake experiments were conducted with6–7-week-old tobacco (Nicotiana tabacum L. cv. Ky 14)seedlings to determine absorption interactions between K⁺ andNH₄⁺. At equal solution concentrations (0.5 mol m^–3) netK⁺ uptake was inhibited 30–35% by NH₄⁺ and NH₄⁺ uptakewas decreased 9–24%. Removal of NH₄⁺ resulted in completerecovery in K⁺ uptake rate, but NH⁴⁺ uptake rate did not recoverwhen K⁺ was removed. In both cases, inhibition of the uptakerate of one cation saturated as the concentration of the othercation was increased up to 0.5 mol m^–3. The relative effectof K⁺-NH₄⁺ interactions was not altered when Cl- was replacedwith SO₄^2–, but the magnitudes of the uptake rates wereless in the absence of Cl-. The V_max for NH₄⁺ uptake was reducedfrom 128 to 105 µmol g^–1 dry wt. h^–1 in thepresence of 0.5 mol m^–3 K⁺ and the K_m for NH₄⁺ doubledfrom 12 to 27 mmol m^–3 in the presence of K⁺. The resultsof these K⁺-NH₄⁺ experiments are interpreted as mixed-noncompetitiveinteractions. However, an enhanced efflux of K⁺ coupled to NH₄⁺influx via an antiporter cannot be ruled out as contributingto the decrease in net K⁺ uptake. Key words: Nicotiana tabacum, K⁺, NH₄⁺, Uptake interactions     

Phosphate uptake and transport in Agrostis capillaris L Effects of non-toxic levels of aluminium and the significance of P and Al speciation

Investigations on the effects of low levels of Al on P adsorption,uptake and translocation in seedlings of the indigenous grassAgrostis capillaris were undertaken. Apparent uptake and transportof H₂ ³²PO₄ from nutrient solutions containing 10 or 100mmolm^–3 phosphate were characterized as functions of timeand concentration. Experiments on ³²P uptake and transport insolutions containing no Al (control) or Al ranging from 3.7to 185 mmol m^–3 at pH ranging from 4.3 to 4.6, showedthat in 10 mmol m^–3 P, effects of Al at 3.7 and 37 mmolm^–3 on the size of the initial uptake shoulder were small,but some increase in subsequent P uptake to the roots was observed,though transport to the shoots was suppressed. With 37 mmolm^–3 Al in nutrient solution containing 100 mmol m^–3P, the uptake shoulder was much increased above the control.Subsequent root uptake was stimulated but transport was unaffected.Lack of toxicity of the Al concentrations used was indicatedby a lack of significant effect on plant fresh weight. AbsorbedAl was almost totally retained in the root in all treatments.Speciation calculations showed that the major species in Alamended nutrient solution at pH 4.4 were H₂PO₄^–, AI³⁺and AIHPO₄⁺, together with substantial amounts of AISO₄⁺ andsoluble aluminium hydroxy complexes (AIOH²⁺, AI(OH)₂⁺), dependingon the relative concentrations of P and Al. The effects of Al,with 10 mmol m^–3 P, on adsorption of complexed P werepartly accounted for in terms of preferential cell wall adsorptionof Al complexes not containing P. Conclusions were drawn aboutthe P-economy of A. capillaris plants growing on soils withlow levels of P and Al. Key words: Phosphorus, aluminium, speciation, Agrostis capillahs L     

Phosphate Uptake in the Cyanobacterium Synechococcus R-2 PCC 7942

Phosphate uptake rates in Synechococcus R-2 in BG-11 media (anitrate-based medium, not phosphate limited) were measured usingcells grown semi-continuously and in continuous culture. Netuptake of phosphate is proportional to external concentration.Growing cells at pH_o 10 have a net uptake rate of about 600pmol m^–2 s^–1 phosphate, but the isotopic flux for³²P phosphate was about 4 nmol m^–2 s^–1. There appearsto be a constitutive over-capacity for phosphate uptake. TheK_m and V_max, of the saturable component were not significantlydifferent at pH_o 7.5 and 10, hence the transport system probablyrecognizes both H₂PO^–₄and HPO^2–₄. The intracellularinorganic phosphate concentration is about 3 to 10 mol m^–3,but there is an intracellular polyphosphate store of about 400mol m^–3. Intracellular inorganic phosphate is 25 to 50kJ mol^–1 from electrochemical equilibrium in both thelight and dark and at pH_o 7.5 and 10. Phosphate uptake is veryslow in the dark ( 100 pmol m^–2 s^–1) and is light-activated(pH_o 7.51.3 nmol m^–2 s^–1, pH_o 10600 pmol m^–2s^–1). Uptake has an irreversible requirement for Mg²⁺in the medium. Uptake in the light is strongly Na⁺-dependent.Phosphate uptake was negatively electrogenic (net negative chargetaken up when transporting phosphate) at pH_o 7.5, but positivelyelectrogenic at pH_o 10. This seems to exclude a sodium motiveforce driven mechanism. An ATP-driven phosphate uptake mechanismneeds to have a stoichiometry of one phosphate taken up perATP (1 PO_{4 in}/ATP) to be thermodynamically possible under allthe conditions tested in the present study. (Received June 16, 1997; Accepted September 4, 1997)     

Induction of Glucose 6-Phosphate Transport Activity in Chloroplasts of Mesembryanthemum crystallinum by the C3-CAM Transition

To study possible changes in the transport metabolites betweenchloroplasts and cytoplasm during CAM induction of Mesembryanthemumcrystallinum, we compared substrate specificity of P1¹ translocator(s)in isolated chloroplasts from the C₃ and CAM-induced plants.The [¹⁴C]glu-cose 6-phosphate (G6P) transport activity was significantonly in the chloroplasts of CAM-mode plants and not detectablein those of C₃-mode, while a similar high rate of [³²P]P_i uptakewas observed with both types of chloroplasts. Kinetic analysisof G6P uptake in the CAM chloroplasts showed a high V_max [10.6µmol (mg Chl)^–1 h^–1] and a comparatively lowK_m value (0.41 mM); the latter was similar to K_i values of P_i,3-phosphoglycerate and phospho-enolpyruvate, 0.30, 0.34 and0.47 mM, respectively. On the other hand, [32P]Pi uptake inthe CAM chloroplasts was inhibited competitively by G6P witha K_i value (8.4 mM) 20-fold higher than the K_m value for G6Puptake, while that in C₃ chloroplasts was not inhibited at all.These results suggest that a new G6P/P_i, counterexchange mechanismis induced in the chloroplast envelope of CAM-induced M. crystallinumin addition to the ordinary type of P, translocator, that cannottransport G6P, already present in the C₃-type chloroplasts. (Received March 17, 1997; Accepted May 10, 1997)     

Active Uptake of CO2 by the Diatom Navicula pelliculosa

Mass spectrometry has been used to investigate the transportof CO₂ in the freshwater diatom Navicula pelliculosa. The timecourseof CO₂ formation in the dark after addition of 100 mmol m^–3dissolved inorganic carbon (DIC) to cell suspensions showedthat no external carbonic anhydrase (CA) was present in thesecells. Upon illumination, cells pre-incubated at pH 75 with100 mmol m^–3 DIC, removed almost all free CO₂ from themedium at an initial rate of 285 µmol CO₂ mg^–1Chl h^–1. Equilibrium between HCO₃^– and CO₂ in themedium occurred rapidly upon addition of bovine CA, showingthat CO₂ depletion resulted from a selective uptake of CO₂ ratherthan an uptake of all inorganic carbon species. However, photosyntheticO₂ evolution rate remained constant after CO₂ had been depletedfrom the medium indicating that photosynthesis is sustainedprimarily by active HCO₃^– uptake. Treatment of cells with2-iodoacetamide (83 mol m^–3) completely inhibited CO₂fixation but had little effect on CO₂ transport since initialrates of CO₂ depletion were about 81% that of untreated cells.Transfer of iodoacetamide-treated cells to the dark caused arapid increase in the CO₂ concentration in the medium largelydue to the efflux of the unfixed intracellular DIC pool whichwas found to be about 194 times the concentration of that inthe external medium. These results indicate that Navicula pelliculosaactively takes up molecular CO₂ against a concentration gradientby a process distinct from HCO₃^– transport. Key words: Dissolved inorganic carbon, carbonic anhydrase, bicarbonate transport, CO₂ transport, mass spectrometry     

Pyruvate Uptake by Mesophyll and Bundle Sheath Chloroplasts of a C4 Plant, Panicum miliaceum L.

Intact chloroplasts were isolated from mesophyll and bundlesheath protoplasts of a C₄ plant, Panicum miliaceum L., to measurethe uptake of [1-¹⁴C]pyruvate into their sorbitol-impermeablespaces at 4?C by the silicone oil filtering centrifugation method.When incubated in the dark, both chloroplasts showed similarslow kinetics of pyruvate uptake, and the equilibrium internalconcentrations were almost equal to the external levels. Whenincubated in the light, only mesophyll chloroplasts showed remarkableenhancement of the uptake, the internal concentration reaching10–30 times of the external level after 5 min incubation.The initial uptake rate of the mesophyll chloroplasts was enhancedabout ten fold by light and was saturated with increasing pyruvateconcentration; K_m and V_max were 0.2–0.4 mM and 20–40µmol(mg Chl)^–1 h^–1, respectively. The lightenhancement was abolished by DCMU and uncoupling reagents suchas carbonylcyanide-m-chlorophenylhydrazone and nigericin. Theseresults indicate the existence of a light-dependent pyruvatetransport system in the envelope of mesophyll chloroplasts ofP. miliaceum. The uptake activity of mesophyll chloroplastsboth in the light and the dark was inhibited by sulfhydryl reagentssuch as mersalyl and p-chloromercuriphenylsulfonate, but thebundle sheath activity was insensitive to the reagents. Thesefindings are further evidence for the differentiation of mesophylland bundle sheath chloroplasts of a C₄ plant with respect tometabolite transport. (Received July 3, 1986; Accepted October 8, 1986)     

Modifying Effects of Non-toxic Levels of Aluminium on the Uptake and Transport of Phosphate in Ryegrass

Apparent uptake and transport of H₂³²PO₄^– from nutrientsolutions containing 100 mmol m^–3 phosphate were characterizedasfunctions of time, concentration and pH in ryegrass seedlings.On a log/log plot, concentration versus uptake to the root resolvedintotwo linear phases, suggesting a change in uptake mechanism orefflux at the break. These results were compared with thosefor ³²P uptake and transport in solutions containing Al rangingfrom 0–185 mmol m^–3. Al addition depressed pH, butbecauseuptake of P was unaffected by pH below 5–0, noadjustments were attempted. Uptake time-courses revealed clearlythe usualinitial adsorption shoulder in the uptake curve, increasingwith Al concentration up to 37 mmol m^–3. Beyond about2 h, P uptaketo the root became linear, at rates increasingwith external Al concentration up to 37 mmol m^–3. Concentrationsof Al muchabove 100 mmol m^–3 were toxic. Al treatmentsdid not affect P transport to the shoot and absorbed Al wasconfined to the root.The quantities of P and Al taken up intothe root indicated storage in cortex cell vacuoles, lockingup significant amounts of P.Experiments with tillering plantsshowed similar characteristics to those with seedlings. Sequesteringof P with Al within the rootcortex cells was evident, particularlyin plants which had been grown in nutrient containing Al fromsoon after germination. Aland P solution chemistry is discussedin the context of this work and the consequences of effectson P uptake for the economy ofphosphate poor upland soils wereconsidered. Key words: Phosphate, aluminium, adsorption, uptake, Lolium perenne L     

Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO₃^– uptake and transport and the effectsof NH₄⁺ or L-glutamate on these processes were investigatedwith excised non-mycorrhizal beech (Fagus sylvatica L.) roots.NO₃^– net uptake followed uniphasic Michaelis-Menten kineticsin a concentration range of 10µM to 1 mM with an apparentK_m of 9.2 µM and a V_max of 366 nmol g^–1 FW h^–1.NH₄⁺, when present in excess to NO₃^–, or 10 mM L-glutamateinhibited the net uptake of NO₃^– Apparently, part of NO₃^–taken up was loaded into the xylem. Relative xylem loading ofNO₃^– ranged from 3.21.6 to 6.45.1% of NO₃^– netuptake. It was not affected by treatment with NH₄⁺ or L-glutamate.¹⁶N/¹³N double labelling experiments showed that NO₃^–efflux from roots increased with increasing influx of NO₃^–and, therefore, declined if influx was reduced by NH₄⁺ or L-glutamateexposure. From these results it is concluded that NO₃^–net uptake by non-mycorrhizal beech roots is reduced by NH₄⁺or L-glutamate at the level of influx and not at the level ofefflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae     

Characteristics of L-lactic acid transport in basal membrane vesicles of human placental syncytiotrophoblast

Thecharacteristics of L-lactic acid transport across thetrophoblast basal membrane were investigated and compared with those across the brush-border membrane by using membrane vesicles isolated from human placenta. The uptake ofL-[¹⁴C]lactic acid into basal membranevesicles was Na⁺ independent, and an uphill transport wasobserved in the presence of a pH gradient([H⁺]_out > [H⁺]_in).L-[¹⁴C]lactic acid uptake exhibitedsaturation kinetics with a K_m value of 5.89 ± 0.68 mM in the presence of a pH gradient.p-Chloromercuribenzenesulfonate and-cyano-4-hydroxycinnamate inhibited the initial uptake, whereas phloretin or 4,4'-diisothiocyanostilbene-2,2'-disulfonate did not.Mono- and dicarboxylic acids suppressed the initial uptake. Inconclusion, L-lactic acid transport in the basal membraneis H⁺ dependent and Na⁺ independent, as is alsothe case for the brush-border membrane transport, and itscharacteristics resemble those of monocarboxylic acid transporters.However, there were several differences in the effects of inhibitorsbetween basal and brush-border membrane vesicles, suggesting that thetransporter(s) involved in L-lactic acid transport in thebasal membrane of placental trophoblast may differ from those in thebrush-border membrane.

     

The Effect of Sucrose Supply on the Energization of Amino Acid Uptake by Cotyledons of Euphorbia lathyris L

The cotyledons of Euphorbia lathyris L. take up sucrose andamino acids from the endosperm. The interaction between theuptake of sucrose and that of amino acids by cotyledons of intactseedlings was investigated. Sucrose (100 mol m^–3) reducedvaline uptake to 75% of the control rate; the active uptakecomponent of valine uptake was reduced from 45 to 25 % of thetotal uptake rate. In a reverse experiment, 100 mol m^–3valine inhibited sucrose uptake by 25%. At 500 mol m^–3sucrose, valine uptake was completely restored to the controlrate, whereas high valine concentrations failed to restore sucroseuptake. The stimulation of valine uptake by sucrose is linkedto the role of sucrose as a primary respiratory substrate. Whenthe cotyledons were bathed in sucrose concentrations rangingfrom 0 to 100 mol m^–3 (these concentrations are non-saturatingwith respect to sucrose uptake), a constant 1.8% of the sucrosetaken up was respired. The K_m of the concentration-dependentsucrose oxidation (44±6 mol m^–3) agreed reasonablywell with that for sucrose uptake (29±6 mol m^–3).When the external sucrose concentration was increased from 100to 600 mol m^–3, the sucrose uptake increased by 30% again,while sucrose oxidation was increased by 300%. This increasewas not due to an increased engagement of the alternative (cyanide-resistant)pathway for respiration. Alternative pathway, Euphorbia lathyris L., fermentation, seedling, sucrose uptake, valine uptake     

The Relationship Between Growth and Oxygen Uptake in Hypoxic Rice Seedlings

Atwell, B. J. and Green way, H. 1987. The relationship betweengrowth and oxygen uptake in hypoxic rice seedlings.—J.exp. Bot. 38: 454–465. Rice seedlings (Oryza saliva L.) were grown in the dark forup to 4 d in solutions containing various concentrations ofO₂. Compared with seedlings grown at 0·250 mol O₂ m^–3,the dry weight of the growing seedling was 14% lower at 0·110mol O₂ m^–3 and 60% lower at 0 mol O₂ m^–3. Decreasesin fresh weight were similar but not identical to decreasesin dry weight, possibly because leaf growth was suppressed evenabove 0·110 mol O₂ m^–3. Oxygen deficiency inhibitedroot growth more severely than coleoptile growth. Coleoptiles from seedlings grown in aerated solution were exposedto an atmosphere of pure N₂ for 30 min. Anoxia caused a declinein ATP content and energy charge, suggestive of decreased oxidativephosphorylation. It is not clear whether the decline in oxidativephosphorylation was solely responsible for impaired growth inhypoxia. In seedlings growing at O₂ concentrations less than 0·110mol O₂ m^–3, significant amounts of ethanol were synthesized.The rate of O₂ uptake decreased markedly below 0·06 molO₂ m^–3; this was presumably near the external O₂ concentrationat which oxidative phosphorylation became limited by the supplyof O₂. The stage of development of the seedlings appeared toinfluence O₂ uptake, possibly through changes in conductanceof the tissue to O₂. Uncouplers were used to confirm that thecritical O₂ concentration was dependent on O₂ diffusion ratherthan enzyme kinetics. Impaired growth above 0·110 molO₂ m^–3 may have been due to a decreased activity of oxygenasesof relatively low affinity for O₂, which in turn altered cellmetabolism. Key words: Growth, oxygen uptake, rice seedlings, hypoxia     

Characterization of inorganic phosphate transport in osteoclast-like cells

Osteoclasts possess inorganic phosphate (P_i) transport systems to take up external P_i during bone resorption. In the present study, we characterized P_i transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-B ligand (RANKL). In undifferentiated RAW264.7 cells, P_i transport into the cells was Na⁺ dependent, but after treatment with RANKL, Na⁺-independent P_i transport was significantly increased. In addition, compared with neutral pH, the activity of the Na⁺-independent P_i transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na⁺-independent system consisted of two components with K_m of 0.35 mM and 7.5 mM. The inhibitors of P_i transport, phosphonoformic acid, and arsenate substantially decreased P_i transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K⁺ ionophore, valinomycin, significantly suppressed P_i transport activity. Analysis of BCECF fluorescence indicated that P_i transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K⁺ ion stimulated P_i transport, suggesting that membrane voltage is involved in the regulation of P_i transport activity. Finally, bone particles significantly increased Na⁺-independent P_i transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a P_i transport system with characteristics that are different from those of other Na⁺-dependent P_i transporters. We conclude that stimulation of P_i transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment. Na⁺-dependent phosphate cotransporter; RAW264.7; phosphate uptake     

The Transport and Metabolism of Urea in Chara australis: II. UREASE AND THE DISTRIBUTION OF TRANSPORTED UREA

The ureolytic enzyme in Chara was investigated. This enzymewas shown to be a urease with an unusually high affinity forurea(K_m = 158 mmol m^-3). Little inhibition of urease activitywas found when intact Chara cells were exposed to the ureaseinhibitors hydroxyurea, acetohydroxamic acid and N-ethylmaleimide,although there was some inhibition of urea uptake. The distribution of radioactivity amongst the amino acid, organicacid and sugar/neutral fractions, determined by ion-exchangechromatography, was very similar whether the Chara internodeswere exposed to ¹⁴C-urea or to H¹⁴CO₃. This suggests that thefraction of the urea-carbon liberated by the urease as CO₂ andretained by the cell is used in photosynthetic carbon-fixation.During the initial 15 min of ¹⁴C-urea uptake, label appearsin the vacuole only in the form of unmetabolized urea. Afterthis time a variety of labelled compounds appear in the vacuole,presumably reflecting the gradual movement of carbon-fixationproducts from the chloroplasts to the cytoplasm and thence intothe vacuole. Key words: Urea transport, metabolism, Chara, urease     

The Effect of Current Photosynthesis on the Origin of Translocates in Old Tomato Leaves

The rate of carbon transport from an old tomato leaf (54 days),grown at 80 W m^–2, was measured under light flux densitiesbetween 7 and 90 W m^–2. Under low light, the rate of carbontransport over a 6 h period was about 1 mg C dm^–2 h^–1,well in excess of the concurrent photosynthetic rate. The lossfrom these leaves of ¹⁴C-leaf assimilate which was fixed beforethe experimental period amounted to 62 per cent of the totalinitial uptake and was higher than that from leaves with higherconcurrent photosynthetic rates. The higher loss of ¹⁴C fromleaves with low photosynthetic rates was due to a greater contributionof ¹⁴C from the starch and residue fractions. The rate of transportappeared to be determined by the concentration of the labilesucrose, not the total sucrose concentration. In comparisonwith young fully-expanded tomato leaves (Ho, 1976) the sizeof the labile sucrose pool appeared to decrease with age. Thephotosynthesistranslocation coefficient was low (k₁k₂=0•21)for an old tomato leaf. Based on these results a scheme of carbonpartitioning in relation to translocation is proposed. Criteriafor assessing the efficiency of translocation in leaves arediscussed.     

Are Mistletoes Shade Plants? CO2Assimilation and Chlorophyll Fluorescence of Temperate Mistletoes and their Hosts

Mistletoes usually have slower rates of photosynthesis thantheir hosts. This study examines CO₂assimilation, chlorophyllfluorescence and the chlorophyll content of temperate host–parasitepairs (nine hosts parasitized by Ileostylus micranthus and Carpodetusserratus parasitized by Tupeia antarctica). The hosts of I.micranthus had higher mean annual CO₂assimilation (3.59 ±0.41 µmol m^-2 s^-1) than I. micranthus(2.42 ± 0.20µmol m^-2 s^-1), and C. serratus(2.41 ± 0.43 µmolm^-2 s^-1) showed higher CO₂assimilation than T. antarctica(0.67± 0.64 µmol m^-2 s^-1). Hosts saturated at significantlyhigher electron transport rates (ETR) and light levels thanmistletoes. The positive relationship between CO₂assimilationand electron transport suggests that the lower CO₂assimilationrates in mistletoes are a consequence of lower electron transportrates. When photosynthetic rates, ETR and chlorophyll a /b ratioswere adjusted for photosynthetically active radiation, hostsdid not have significantly higher CO₂assimilation (3.21 ±0.37 µmol m^-2 s^-1) than mistletoes (2.54 ± 0.41µmol m^-2 s^-1), but still had significantly higher ETRand chlorophyll a / b ratios. The electron transport rates,saturating light and chlorophyll a / b ratios of sun leavesfrom mistletoes were similar to host shade leaves. These responsesindicate that in comparison with their hosts, mistletoe leaveshave the photosynthetic characteristics of the leaves of shadeplants. Copyright 2000 Annals of Botany Company CO₂assimilation, photosynthetic active radiation (PAR), chlorophyll fluorescence, electron transport rate (ETR), photochemical quenching (q_p), non-photochemical quenching (q_n), sun and shade leaves, chlorophyll content, Ileostylus micranthus, Tupeia antarctica, New Zealand     

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2

The general phosphate need in mammalian cells is accommodated by members of the P_i transport (PiT) family (SLC20), which use either Na⁺ or H⁺ to mediate inorganic phosphate (P_i) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na⁺-dependent P_i (NaP_i) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with ³²P_i as a traceable P_i source. For PiT1, the Michaelis-Menten constant for P_i was determined as 322.5 ± 124.5 µM. PiT2 was analyzed for the first time and showed positive cooperativity in P_i uptake with a half-maximal activity constant for P_i of 163.5 ± 39.8 µM. PiT1- and PiT2-mediated Na⁺-dependent P_i uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na⁺ dependency patterns. However, only PiT2 was capable of Na⁺-independent P_i transport at acidic pH. Study of the impact of divalent cations Ca²⁺ and Mg²⁺ revealed that Ca²⁺ was important, but not critical, for NaP_i transport function of PiT proteins. To gain insight into the NaP_i cotransport function, we analyzed PiT2 and a PiT2 P_i transport knockout mutant using ²²Na⁺ as a traceable Na⁺ source. Na⁺ was transported by PiT2 even without P_i in the uptake medium and also when P_i transport function was knocked out. This is the first time decoupling of P_i from Na⁺ transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E⁵⁵ and E⁵⁷⁵ are responsible for linking P_i import to Na⁺ transport in PiT2. inorganic phosphate transport; retroviral receptor; SLC20