31P NMR Measurements of the Cytoplasmic and Vacuolar Pi Content of Mature Maize Roots: Relationships with Phosphorus Status and Phosphate Fluxes

The vacuolar and cytoplasmic inorganic phosphate (Pi) contentof the mature regions of maize roots was measured by a ³¹P NMRtechnique which used an external standard to avoid the needfor tissue extraction and which exploited the relatively rapidrelaxation of cytoplasmic Pi in order to improve the detectionof this pool in fully-vacuolated cells. In mature roots of maize growing with abundant external phosphate,the concentration of Pi in the cytoplasm was approximately 6.5mol m^–3. When these plants were deprived of external phosphate,the vacuolar Pi content of the roots decreased rapidly, butthe cytoplasmic Pi concentration initially remained constantand did not begin to decline until P-stress became severe. Calculationsshow that withdrawal of Pi from the vacuoles into the cytoplasmunder these conditions would be against an electrochemical gradient. During P-starvation, an increased capacity for Pi influx developed,preceding any detectable change in the cytoplasmic Pi contentof the roots. This response is considered in terms of paralleleffects on transport sites for phosphate at the plasmalemmaand at the tonoplast. Comparisons of simultaneous rates of influxand net uptake implied that phosphate efflux accounted for <10% of influx in plants of a steady or declining P-status. However,direct measurements of efflux suggested that this process maybe temporarily accelerated when plants are recovering from P-stress. Key words: P-nutrition, subcellular compartmentation     

Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Interspecific and intraspecific mitochondria-induced cytoplasmic transformation in yeasts

Spheroplasts from respiratory-deficient (rho^–) yeastswere transformed into rho⁺ cells by incubation with mitochondriaprepared from respiratory-sufficient (rho+) yeasts in the presenceof polyethylene glycol 4000 and CaCl₂. Spheroplasts of buddingyeast rho^– strains were also transformed into rho⁺ cellsby treatment with mitochondria prepared from heterospecificbudding or fission yeast strains. All the transformed regeneratantsyielded rho⁺ yeast colonies which bore chromosomal genetic characteristicsof the spheroplasts used and cytoplasmic genetic characteristicsof the mitochondria used. These indicate that mitochondrialgenes or mitochondria themselves introduced by the incubationfunctioned normally in the rho^– cells regardless of thespecies difference of the recipient spheroplasts. ¹A preliminary report appeared in ref. 17. (Received October 31, 1978; )     

Electron Donation from Cyclic and Respiratory Flows to the Photosynthetic Intersystem Chain is Mediated by Pyridine Nucleotide Dehydrogenase in the Cyanobacterium Synechocystis PCC 6803

The redox kinetics of P700 induced by far-red light and a pulseof strong white light in wild type cells were compared withthose in NAD(P)H dehydrogenase (NDH)-defective mutants of thecyanobacterium Synechocystis PCC 6803. The wild type cells showedthe electron donation from the respiratory donor and the photoreductantgenerated in PS I to P700⁺ through the plastoquinone, whichis mediated by a Hg²⁺-sensitive enzyme. The NDH-defective mutantcells, however, did not show the electron transfer to P700⁺through the plastoquinone from both the photoreductant in PSI and cytosolic electron donors using pyndine nucleotides asan intermediate. Thus, NDH appears to be the site of main entryof electrons into the plastoquinone pool in the NAD(P)H-mediatedcyclic electron flow and the respiratory electron flow in Synechocystis. (Received August 31, 1992; Accepted October 1, 1992)     

A 31P-NMR Study of the Uptake and Compartmentation of Manganese by Maize Roots

The uptake and compartmentation of manganese by maize roots,from solutions containing between 1 µM and 1 mM Mn²⁺,was monitored in vivo by ³¹P nuclear magnetic resonance (NMR)spectroscopy. Qualitatively, NMR provided a convenient methodfor observing the effects of pH, anoxia, metabolic inhibitors,and competition with magnesium on the uptake of manganese andthe resultshighlighted the role of the vacuole as a sink forMn²⁺. Quantitatively, it was established thatroot tissues couldmaintain a low concentration of free Mn²⁺ in the cytoplasm duringmanganese uptake and that there is a non-equilibrium distributionof Mn²⁺ between the cytoplasm and the vacuole. Typically exposureto Mn²⁺ in the range 10–100 µM resulted in a submicromolarpool of Mn²⁺ in the cytoplasm and a vacuolar pool of 10 µMand it was concluded that the movement of Mn²⁺ out of the cytoplasmmust be energy consuming. Overall the results draw attentionto the similarity between the subcellular distribution of manganeseand calcium and provide some support for the suggestion thatmanganese, like calcium, might have a control function in normalcells. Key words: Cytoplasm, intracellular compartmentation, manganese, ³¹P-NMR, vacuole     

Oxidative ATP synthesis in skeletal muscle is controlled by substrate feedback

Data from ³¹P-nuclear magnetic resonance spectroscopy of human forearm flexor muscle were analyzed based on a previously developed model of mitochondrial oxidative phosphorylation (PLoS Comp Bio 1: e36, 2005) to test the hypothesis that substrate level (concentrations of ADP and inorganic phosphate) represents the primary signal governing the rate of mitochondrial ATP synthesis and maintaining the cellular ATP hydrolysis potential in skeletal muscle. Model-based predictions of cytoplasmic concentrations of phosphate metabolites (ATP, ADP, and P_i) matched data obtained from 20 healthy volunteers and indicated that as work rate is varied from rest to submaximal exercise commensurate increases in the rate of mitochondrial ATP synthesis are effected by changes in concentrations of available ADP and P_i. Additional data from patients with a defect of complex I of the respiratory chain and a patient with a deficiency in the mitochondrial adenine nucleotide translocase were also predicted the by the model by making the appropriate adjustments to the activities of the affected proteins associates with the defects, providing both further validation of the biophysical model of the control of oxidative phosphorylation and insight into the impact of these diseases on the ability of the cell to maintain its energetic state. computational model; mitochondria; cellular energetics; oxidative phosphorylation; ³¹P-NMR spectroscopy     

Rapid Cytoplasmic Alkalization and Dynamics of Intracellular Compartmentation of Inorganic Phosphate during Adaptation against Salt Stress in a Halotolerant Unicellular Green Alga Dunaliella tertiolecta: 31P-Nuclear Magnetic Resonance Study

By ³¹P-in vivo nuclear magnetic resonance spectroscopy, twointracellular compartments were detected, and their pH valueswere estimated in intact cells of a halotolerant unicellulargreen alga Dunaliella tertiolecta. They were identified as thecytoplasm (pH 7.1) and vacuoles (pH 6.0). Vacuoles were alsovisualized with a fluorescence-differential interference microscope.During the adaptation to the salt stress (NaCl concentrationfrom 0.17 to 1.0 M) where cells rapidly synthesize glycerolas osmoticum, both cytoplasmic and vacuolar pH showed transientincreases to about 8 and 6.5, respectively. Subsequently, cytoplasmicinorganic phosphate level, as well as sugar phosphates and terminalphosphate group levels increased. These drastic changes in chemicalenvironment in the cytoplasm including pH and inorganic phosphateconcentration are discussed to be key factors for osmoregulationthat activate the synthesis and inhibit the breakdown of glycerol. (Received August 22, 1988; Accepted February 6, 1989)     

31P-NMR DIFFERENTIATION BETWEEN INTRACELLULAR PHOSPHATE POOLS IN COSMARIUM (CHLOROPHYTA)1

³¹P nuclear magnetic resonance (NMR) spectroscopy of intact Cosmarium sp. cells is presented as a suitable tool for the differentiation of intracellular accumulation pools of polyphosphates. The cold trichloroacetic acid (TCA) insoluble fraction is shown to contain most of the total cellular phosphate in the phosphate rich Cosmarium cells. Moreover, evidence from a ³¹ P-NMR study and electron microscopic observations of cold TCA treated Cosmarium cells indicate that this fraction consists mostly of polyphosphates which seem to retain the native morphological structure observed in the untreated cells. The determination of orthophosphate in the hot water extract of Cosmarium cells did not measure the polyphosphate pools. Determination of total phosphorus content in the hot water extract rendered a value three times higher than the frequently used orthophosphate determination procedure. However, as revealed by the ³¹P-NMR spectra and the chemical analyses of the extract and of the treated cells, even total phosphorus in the extract measured only 30% of the total cellular phosphorus. ³¹P-NMR enabled the unequivocal chemical identification of the major phosphate compounds in the hot water extract (“Surplus P”) as orthophosphate and polyphosphates of about 10 phosphate units chainlength. More than 70% of the accumulation pool of polyphosphates was still in the cells after extraction. However, the electron microscopy study revealed that the native granular structure of polyphosphates had been destroyed by the hot water extraction procedure.     

Pyridine and Adenine Nucleotide Status, and Pool Sizes of a Range of Metabolites in Chloroplasts, Mitochondria and the Cytosol/Vacuole of Avena Mesophyll Protoplasts during Dark/Light Transition: Effect of Pyridoxal Phosphate

Leaf mesophyll protoplasts of Avena sativa L. underwent dark/lighttransition in the absence or presence of pyridoxal phosphate(PLP), a specific inhibitor of the phosphate translocator ofchloroplasts. By combining rapid fractionation of protoplastswith enzymatic cycling, the contents of metabolites (adeninenucleotides, pyridine nucleotides, triose phosphates, 3-phosphoglycerate,inorganic phosphate, aspartate, malate, oxaloacetate, glutamate,2-oxoglutarate and citrate) associated with the chloroplasts,mitochondria and cytosol/vacuole were determined. Fluctuationsof metabolite pools occurred in all compartments on illumination.Mitochondria showed immediate inhibition of their tricarboxylicacid cycle activities, as indicated by a transiently increasedNADH/NAD⁺ ratio and elevated malate contents within 60 s ofillumination. During this period large transient increases intriose phosphates took place in all fractions. Incubation of intact protoplasts with PLP severely affectedthe compartmented pool sizes, producing the typical patternof inhibition of the chloroplast phosphate translocator. Themitochondrial pool sizes of the metabolites responded to light,if at all, differently than did the controls. ¹ Dedicated to Prof. Hubert Ziegler on the occasion of his 60thbirthday. (Received July 23, 1984; Accepted October 19, 1984)     

Changes in the Cytoplasmic and Vacuolar pH in Intact Cells of Mung Bean Root-Tips under High-NaCl Stress at Different External Concentrations of Ca2+ Ions

The cytoplasmic pH and the vacuolar pH in root-tip cells ofintact mung bean seedlings under high-NaCl stress were measuredby in vivo ³¹P-nuclear magnetic resonance (³¹P-NMR) spectroscopy.When roots were incubated with high levels (100 mM) of NaClat the control external concentration (0.5 mM) of Ca²⁺ ions,the vacuolar pH increased rapidly from 5.6 to 6.2 within 3 h,while the cytoplasmic pH only decreased by a mere 0.1 pH uniteven after a 24-h incubation under high-NaCl conditions. Theincrease in vacuolar pH induced by the high-NaCl stress wasdiminished by an increase in the external concentration of Ca²⁺ions from 0.5 mM to 5 mM. The intracellular concentration ofNa⁺ ions in the root-tip cells increased dramatically upon perfusionof the root cells with 100 mM NaCl, and high external levelsof Ca²⁺ ions also suppressed the in flow of Na⁺ ions into thecells. The vacuolar alkalization observed in salt-stressed rootsmay be related to the inhibition of an H⁺-translocating pyrophosphatasein the tonoplast, caused by the increase in the cytoplasmicconcentration of Na⁺ ions. It is suggested that, although thevacuolar pH increased markedly under salt stress, the cytoplasmicpH was tightly regulated by some unidentified mechanisms, suchas stimulation of the H⁺-translocating ATPase of the plasmalemma,in roots of mung bean under salt stress. (Received April 18, 1992; Accepted July 6, 1992)     

Metabolic changes of the Antarctic green alga Prasiola crispa subjected to water stress investigated by in vivo 31P NMR

The energy status and the phosphate metabolism of Prasiola crispduring and after desiccation stress was investigated by in vivo³¹P NMR. The effect of desiccation was simulated by additionof the nonionic osmoticum PEG 200 (polyethylene glycol). Photosynthesisand respiration were effectively inhibited under these conditions.The most notable changes in the in vivo ³¹P NMR spectra werean increase in the cytoplasmic inorganic phosphate signal afterPEG stress, a decrease in the polyphosphates and a lowfieldshift of the core polyphosphate signal followed by an appearanceof extracellular inorganic phosphate. Cytoplasmic pH remainedalmost constant during stress. After a return to control conditions,photosynthesis and respiration recovered within 4 h as wellas the concentrations of the phosphorus metabolites. An as yetunassigned phosphate signal increased in the phosphodiesterregion of the NMR spectra. Simultaneousty, the polyphosphatesignal recovered in intensity and chemical shift. It is suggestedthat phosphate metabolism and complexation of cations to polyphosphatesmay play an important role in the distinct desiccation toleranceof P. crispa. Key words: In vivo ³¹P NMR, Prasiola crispa, desiccation tolerance, polyphosphates     

Sodium-Stimulation of Phosphate Uptake in the Cyanobacterium Anabaena PCC 7119

Anabaena PCC 7119 showed higher rates of phosphate uptake whencells were under P-starvation. Phosphate uptake was energy-dependentas indicated the decrease observed when assays were performedin the dark or in the presence of inhibitors of photosyntheticelectron transport, energy transfer and adenosine triphosphataseactivity. Phosphate uptake was stimulated by Na⁺ both in P-sufficientcells and P-starved cells. Li⁺ and K⁺ acted as partial analoguesfor Na⁺. The Na⁺-stimulation of phosphate uptake followed Michaelis-Mentenkinetics, half-saturation (K) of phosphate uptake was reachedwith a Na⁺ concentration of 212 µM. The absence of Na⁺reduced the rates of phosphate uptake at all phosphate concentrationsassayed (1–20 µM). The maximum uptake rates (V_max)decreased from 658 nmol P (mg dry wt)^-1 h^-1 in the presenceof Na⁺ to 149 nmol P (mg dry wt)^-1 h^-1 in the absence of Na⁺.The absence of Na⁺ did not change significantly the concentrationof phosphate required to reach half-saturation (K) (3.01 µMin the presence of Na⁺ vs 3.21 µM in the absence of Na⁺).In the presence of Na⁺ the rate of phosphate uptake was affectedby the pH; optimal rates were observed at pH 8. In the absenceof Na⁺ phosphate uptake was not affected by the pH; low rateswere observed in all cases. Monensin, an ionophore which collapsesNa⁺-gradients, reduced the rate of phosphate uptake in Na⁺-supplementedcells. These results indicated the existence of a Na⁺-dependentphosphate uptake in Anabaena PCC 7119. (Received September 8, 1992; Accepted November 17, 1992)     

Quantitative Aspects of the 31P-NMR Detection of Manganese in Plant Tissues12

The uptake of manganese by maize roots was monitored in vivoby ³¹P nuclear magnetic resonance (NMR) spectroscopy and a quantitativeanalysis was developed on the basis of the line broadening ofthe vacuolar orthophosphate (P₁) signal. The line broadening,which was followed indirectly by measuring changes in the reciprocalpeak height of the P₁ signalin fully relaxed spectra, was foundto depend on pH and P₁ concentration, as well ason the presenceof organic acids, but for P₁ concentrations in the millimolarrange the method was sensitive to Mn₂₊ concentrations as lowas 0·1–1 µM. A linear relation was establishedbetween the reciprocal peak height of the vacuolar P₁ signalobserved in vivo and the total manganese content of the tissuedetermined subsequently by atomicabsorption. However, the paramagneticcontribution to the line widthobserved in vivo was much smallerthan expected from measurements on simple solutions and freeze-thawextracts and it was concluded that less than 5% of the manganesetaken up by the root tissue was present in the vacuoles as solubleMn²⁺. The ability to detect the free pool of divalent manganeseis one of several advantages of the ³¹P-NMR method relativeto the analogous¹H-NMR method based on the interaction betweenmanganese and water; and the non-invasive nature ofthe method,coupled with the potential to distinguish the cytoplasmic andvacuolar manganese fractions, allows the NMR method to complementthe information obtained by atomic absorption. Key words: Cytoplasm, intracellular compartmentation, manganese, ³¹P-NMR, vacuole     

Subcellular Distribution of Inorganic Phosphate, and Levels of Nucleoside Triphosphate, in Mature Maize Roots at Low External Phosphate Concentrations: Measurements with 31P-NMR

Maize plants were grown in nutrient solution without phosphate,or in which inorganic phosphate (Pi) was maintained at nearlyconstant concentrations of 1 µM, 10µM or 0·5mM. In vivo ³¹P-NMR measurements showed that there was no discernibledifference in the cytoplasmic Pi content (µmol cm^–3root volume) of the mature roots of plants exposed to 1 µM,10µM or 0·5 mM external phosphate for up to 12d. However, the vacuolar Pi content of the mature roots variedabout 10-fold between these three groups. The cytoplasmic Pi content of roots receiving no external phosphatedecreased significantly after about 7 d total growth, and atabout this time the vacuolar pool of Pi became too small foraccurate measurement. The presence of 1 µM Pi in the nutrientsolution completely prevented this decline in cytoplasmic Pi,and there was some evidence that it also raised the Pi contentof the root vacuoles above the almost undetectable level foundin the totally P-starved roots. During the first 7–9 d of growth, the nucleoside triphosphatecontent of the mature roots was unaffected by the concentrationof phosphate in the nutrient solution. The results highlight the close control of cytoplasmic concentrationsof certain important phosphorus metabolites in roots growingin soil of normal agricultural fertility. Key words: Vacuole, cytoplasm, intracellular compartmentation, NTP, P-nutrition     

Metabolism and Biosynthesis of Cytokinins in Tobacco Crown Gall Cells

The metabolism of ribosylzeatin (RZ) was studied using tobaccocrown gall cells which produce RZ as one of the major endogenouscytokinins. When [8-¹⁴C]RZ was fed to the cells, it was convertedinto its phosphate (which was rigorously determined to be the5'-monophosphate), RZ-O-glucoside, inosine (or its phosphate),adenosine and adenosine-O-glucoside. When [8-¹⁴C]N⁶-(²-isopentenyl)adenosine(i⁶Ado), a probable precursor of RZ, was fed to the cells, itwas converted into (i⁶Ado)-O-glucoside, inosine (or its phosphate),adenosine, adenosine-O-glucoside and adenosine phosphate, butno incorporation of radioactivity into RZ was observed. Thepresent study led to the following conclusions: i) i⁶Ado isnot a precursor of RZ in the cells, ii) both deaminase and cytokininoxidase are involved in the catabolism of cytokinin, and iii)the metabolism of RZ is quite different from that of i⁶Ado. (Received December 24, 1985; Accepted April 1, 1986)     

Effect of linolenate on photosynthesis by intact spinach chloroplasts II. Influence of preillumination of leaves on the inhibition of photosynthesis by linolenate

The inhibitory effect of linolenate on intact spinach chloroplastsdepends on the level of the internal pool of metabolites. Chloroplastsfrom preilluminated leaves or chloroplasts artificially loadedwith 3-phosphoglyceric acid required higher concentrations oforthophosphate for maximal rates of CO₂ dependent O₂ evolutionthan untreated chloroplasts. The loaded chloroplasts were moresensitive to linolenate, and in the presence of linolenate theoptimal phosphate concentration was shifted toward lower values.We propose that the inhibition of photosynthesis by linolenateis due to inhibition of the "phosphate translocator". ¹ Part of this work has been published in the Book of Abstracts,4th International Congress on Photosynthesis, Reading, U.K.,1977, p. 265–266. ² This work is part of a doctoral programme carried out by L.Mv6 Akamba in this laboratory. ³ To whom reprint requests should be adressed. (Received October 14, 1978; )     

Phosphate Compounds of Scenedesmus C-2A' in Darkness or Blue Light as Measured by 31P NMR

Phosphorus-31 nuclear magnetic resonance (³¹P NMR) spectra ofintact cells of Scenedesmus mutant C-2A' and of their perchloricacid extracts are presented. Sugarphosphates, including glucose-6-phosphateand fructose-1,6-bisphosphate, orthophosphate, nucleotide di-and triphosphates, NAD(P), UDPG and—in the case of intactcells—also polyphosphates were identified. Blue light,which is known to stimulate the carbohydrate breakdown of greenalgae, leads to a transient drop in P_i, a pronounced decreasein the ATP/ADP ratio, and an increase in sugarphosphates, givingrise to the idea that the enhancement of phosphorolytic starchbreakdown is a primary response to blue light. Addition of glucoseto Scenedesmus mutant cells leads to comparable changes (besidesan additionally enhanced glucose-6-phosphate level), which thussupport the view that blue light stimulates dark-type respiration.Altogether the results demonstrate the applicability of ³¹PNMR spectroscopy to the study of the metabolism of green algae. (Received December 7, 1984; Accepted February 12, 1985)     

Inhibition of phosphoglucomutase activity by lithium alters cellular calcium homeostasis and signaling in Saccharomyces cerevisiae

Phosphoglucomutase is a key enzyme of glucose metabolism that interconverts glucose-1-phosphate and glucose-6-phosphate. Loss of the major isoform of phosphoglucomutase in Saccharomyces cerevisiae results in a significant increase in the cellular glucose-1-phosphate-to-glucose-6-phosphate ratio when cells are grown in medium containing galactose as carbon source. This imbalance in glucose metabolites was recently shown to also cause a six- to ninefold increase in cellular Ca²⁺ accumulation. We found that Li⁺ inhibition of phosphoglucomutase causes a similar elevation of total cellular Ca²⁺ and an increase in ⁴⁵Ca²⁺ uptake in a wild-type yeast strain grown in medium containing galactose, but not glucose, as sole carbon source. Li⁺ treatment also reduced the transient elevation of cytosolic Ca²⁺ response that is triggered by exposure to external CaCl₂ or by the addition of galactose to yeast cells starved of a carbon source. Finally, we found that the Ca²⁺ overaccumulation induced by Li⁺ exposure was significantly reduced in a strain lacking the vacuolar Ca²⁺-ATPase Pmc1p. These observations suggest that Li⁺ inhibition of phosphoglucomutase results in an increased glucose-1-phosphate-to-glucose-6-phosphate ratio, which results in an accelerated rate of vacuolar Ca²⁺ uptake via the Ca²⁺-ATPase Pmc1p. calcium influx; calcium signal; galactose; glucose phosphate     

Purification and Characterization of Three Distinct Protein Kinases from Marchantia polymorpha

Three protein kinases (HK-I, HK-II and HK-III) have been partiallypurified from the 1.0 M KC1 extract of Marchantia polymorphaand biochemically characterized. It was found that (i) the molecularweights of HK-I, HK-II and HK-III were approximately 23 kDa,47 kDa and 28 kDa, respectively; (ii) these three kinases requireddivalent cations, such as Mn²⁺ and Mg²⁺, but not Ca²⁺, for activity;and (iii) histone H1 was an effective phosphate acceptor forboth HK-I and HK-II, whereas the other kinase (HK-III) effectivelyphosphorylated whole histone (Type II-A from calf thymus) ratherthan histone H1. Heparin (20µg/ml), an inhibitor of caseinkinase II, significantly stimulated the phosphorylation of cellularpolypeptides by HK-II, which was thermo sensitive even at 30?C,rather than that by the other kinases (HK-I and HK-III). Moreover,experiments in vitro and in vivo to determine the native phosphateacceptors for HK-II indicated that a 60-kDa cellular polypeptidemay be one of the native phosphate acceptors for the proteinkinase. In addition, the similarity in properties of cdc2-kinase,which plays an important role in the cell cycle (in the transitionfrom the G₂ phase to mitosis) of yeast and many eukaryotic cells,to HK-II is discussed. (Received May 2, 1990; Accepted December 6, 1990)     

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)