Untersuchungen über die Raten der Polyphosphatsynthese durch die Photophosphorylierung bei Ankistrodesmus braunii

Summary Short-time experiments with ³²P-labelled phosphate and chase experiments with equally labelled cells were carried out with synchronized algae under conditions of optimum phosphate uptake. In short-time experiments, in the presence as in the absence of CO₂, orthophosphate and organic phosphates are rapidly labelled, but their time curves show saturation behaviour after 10 to 20 min. Labelling of polyphosphates proceeds at a constant rate after a short lag period of about 5 min. In equally labelled algae ³²P-labelling correspondingly decreases in orthophosphate and in organic phosphates, but increases by about the same amount in the fraction of acid-insoluble polyphosphates. In the presence of external phosphate and in the light, polyphosphates show no visible decay within the 20 min of the chase experiments.A comparison of the two kinds of experiments suggests that polyphosphates are secondary products of photophosphorylation following only after orthophosphate and organic phosphates, probably after ATP. The rates of photophosphorylation are certainly much higher than the rates of labelling in organic phosphates because of the limiting phosphate uptake. Since the polyphosphates show no decay during the time of the experiments their turnover is low and the rates of polyphosphate labelling after a phosphate starvation period, and after the short lag period, can be regarded as approximate rates of polyphosphate synthesis. These rates are lower than the rates of phosphate uptake.In young cells of the synchronous culture phosphate replenishment after a 5-h starvation requires 2 to 3 h. After replenishment or in a culture undisturbed by phosphate starvation, the rates of polyphosphate accumulation, like the rates of phosphate uptake are much lower. In the presence of CO₂ they are constant for several hours, if related to culture volume with constant cell number. Polyphosphate accumulation is proportional to phosphate uptake under these conditions amounting to about one third. In the absence of CO₂, the rates decrease after 2 to 4 h of CO₂-starvation and, like in short-time experiments a large proportion of the phosphate taken up is used for polyphosphate accumulation. The low rates of long-time experiments may represent a steady state between formation and decay of polyphosphates. Since the cells kept in the absence of CO₂ are prevented from growing they actually accumulate more polyphosphates per cell volume, per chlorophyll, and per dry weight than the cells in the presence of CO₂.The rates of polyphosphate formation are discussed with respect to their turnover in the light observed by other investigators. They are regarded to be a result of competition for ATP together with the orthophosphate pool of the cells, and of the compartmentation. The rates of polyphosphate formation are rather low compared with the probable rates of ATP formation under various conditions of photophosphorylation. Therefore, the formation of polyphosphates is regarded as a process of secondary order of magnitude in the energy metabolism of algal cells.

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Im Text verwendete Abkürzungen P₁ Trichloressigsäure lösliche Phosphate - davon P_i Orthophosphat - P_o organisches Phosphat - P_ul Hydrolyse-labiles TCE-unlösliches Phosphat - P_us Hydrolyse-stabiles TCE-unlösliches Phosphat - P_ges Gesamtphosphat, bei kurzzeitiger ³²P-Markierung Phosphataufnahme - Chl Chlorophyll     

Die Bildung von Polyphosphaten bei Ankistrodesmus braunii durch Photophosphorylierung im Rotlicht von 683 und 712 nm unter Stickstoffatmosphäre

Summary A simplified extraction procedure was used to study the effect of red and far-red light (683 and 712 nm) on the incorporation of ³²P and on the distribution pattern of various phosphate fractions in 8 min-experiments under nitrogen. Nitrogen was used to avoid competition for energy-rich phosphate bonds between CO₂-fixation and the formation of polyphosphates. The total incorporation of ³²P was higher in red than in far-red light, and so was the percentage of the acidsoluble organic phosphate. In the absence of oxygen the incorporation in the dark was rather low and mainly confined to orthophosphate. The ratio between the amount of labelling of polyphosphates and that of acid-soluble organic phosphates was higher in far-red light and in the dark than in red light.DCMU, even in a nitrogen atmosphere, produced a servre inhibition in red light. This inhibition increased with increasing light intensity. The labelling of organic phosphates was more affected than that of polyphosphates, while orthophosphate incorporation was least inhibited. In far-red light, DCMU exerted little influence except at a rather high light intensity, showing that cyclic photophosphorylation was proceeding alone.Antimycin A, on the other hand, was almost ineffective in strong red light, but produced a serious inhibition in far-red light. In red light of medium intensity, antimycin effected some inhibition, although much less than DCMU. Under these conditions the effect of the two inhibitors was additive when they were applied together. Labelling of polyphosphates was more sensitive to antimycin A than labelling of acid-soluble organic phosphates.It may be concluded from the data presented that far-red light produces conditions for pure cyclic photophosphorylation, whereas a large proportion of the photophosphorylation taking place in red light in the absence of CO₂ and exogenous oxygen might be regarded as pseudocyclic. The distribution pattern of the phosphorylated fractions under the different conditions suggests that polyphosphate formation in the light is favoured but not exclusively effected by cyclic photophosphorylation.

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Herrn Prof. Dr. F. Overbeck zum 70. Geburtstag gewidmet.     

Der Einfluß von CO2 und pH auf die 32P-Markierung von Polyphosphaten und organischen Phosphaten bei Ankistrodesmus braunii im Licht

Summary The effect of CO₂ on the ³²P-labelling of polyphosphates and acid-soluble organic phosphates is studied in synchronously grown cultures of the green alga Ankistrodesmus braunii, using trichloroacetic acid treatment and acid hydrolysis for the fractionation of the phosphorus compounds.Three per cent CO₂ in nitrogen causes an inhibition of the labelling of polyphosphates but a marked increase of ³²P in organic phosphates, whereas oxygen (CO₂-free air) produces the reverse effect. Polyphosphates and ATP are the fractions most stimulated by O₂, while stable organic phosphates show the strongest inhibition. Labelling of nucleic acids is relatively indifferent to both oxygen and CO₂. Three per cent CO₂ in air causes the same distribution of ³²P-labelling as 3 per cent CO₂ in N₂. ³²P-labelling is strongly dependent on the pH of the medium. In the absence of CO₂, polyphosphate labelling is highest in the acidic range, whereas organic phosphates and ATP show optimum labelling and the highest percentage of the total ³²P in the alkaline pH range. The effect of CO₂ is strongest between pH 5 and 6, that of oxygen between pH 8 and 9. Apparently the pH of the medium exerts a considerable influence upon the phosphate metabolism inside the cells.Increasing concentration of CO₂ lead to the same change of ³²P-labelling in nitrogen as in air and to saturation at about 1 per cent CO₂ under the conditions used. The curves are in good agreement with those of O₂-evolution at increasing concentrations of CO₂, but they show completely different rates.Young cells respond to CO₂ and O₂ differently from cells in the photosynthetically most active stage. In young cells both gasses are less effective.The effect of CO₂ is explained by a strong increase in noncyclic photophosphorylation which can proceed only slowly in N₂. ATP-consumption connected with high rates of CO₂-fixation may be the reason for the low rates of ³²P-labelling in the polyphosphate fraction when CO₂ is present. The influence of external pH on ³²P-labelling is partly due to the pH-dependence of phosphate uptake, but the different response of several fractions to the pH of the medium suggests that the pH of the cytoplasm and possibly even the pH of the interior of the chloroplasts is affected by the external pH. The effect of O₂ in the absence of CO₂ or at low CO₂-concentrations is explained by the well-known inhibition of photosynthesis by oxygen. Increasing concentrations of CO₂ reverse this inhibition and correspondingly change the distribution of ³²P between the phosphate fractions. The change in sensitivity to CO₂ and O₂ with the cell age is consistent with the change in the rates of maximum photosynthetic CO₂-fixation.

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Herrn Prof. Dr. W. Schumacher zum 70. Geburtstag gewidmet.     

The Effect of Light Intensity on Total Photophosphorylation and the ATP Level in Scenedesmus

The dependence of in vivo photophosphorylation on light intensity was studied in the unicellular green alga Scenedesmus obtusiusculus. By selective use of the inhibitor DCMU, phosphorylation in (I) the complete system, (II) the pseudocyclic system alone, and (III) the true cyclic system alone, were followed. When the total binding of phosphate was studied, all reaction types became light saturated in about the same manner. The effect of DCMU on the level of ATP varied according to light intensity. As for the specific systems of photophosphorylation, the following ATP data were found: (I) In the complete system the level of ATP decreases with light intensity. (II) Under pseudo-cyclic conditions light first increases and then decreases the ATP level. Under the atmospheric conditions used (i.e. CO₂-free nitrogen) this indicates a regulation between photophosphorylation and glycolysis, for which possible explanations are discussed. (III) In the true cyclic conditions light has little effect on the ATP level. The possibility is indicated that there is a structural difference between the non-cyclic (site 1) and the pseudocyclic (site 2) sites of photophosphorylation on the one hand and the true cyclic site (3) on the other.     

In vivo P-31 NMR measurements of phosphate metabolism in Platymonas subcordiformis as related to external pH

The phosphate metabolism of Platymonas subcordiformis was investigated by 31P-NMR spectroscopy with special attention on the effect of external pH. Glycolyzing cells and cells energized by respiration or photosynthesis gave spectra dependent upon their metabolic state. The transition from deenergized to energized states is accompanied by a shift of cytoplasmic pH from 7.1–7.4, an increase of ATP level and-in well energized cells-the appearance of a new signal tentatively assigned to phosphoarginine.The spectra remain stable over a wide range of external pH. Cytoplasmic pH is well regulated in respiring cells for external pH in the range 5.3–12.3. The typical 0.4 units difference of internal pH in energized as compared to deenergized cells is not affected by external pH in the range 6–12. The intensity of a signal attributed to PEP is markedly increased at high external pH. pH regulation is less efficient below external pH of 6 in deenergized cells. Below pH 3.8 oxidative phosphorylation ceases. Upon raising cytoplasmic pH to 7.4 in deenergized cells polyphosphate chains start to disintegrate.Abbreviations PEP Phosphoenolpyruyate - P _i inorganic phosphate - PP _i inorganic pyrophosphate - poly P polyphosphates - PP-1, PP-2, PP-3 terminal, second, and third phosphate residue of polyphosphates - PP-4 core phosphate residues of polyphosphates - pH _i , pH _o internal (cytoplasmic) and external pH - NTP/NDP nucleotide triphosphate/-diphosphate - S/N signal to noise ratio     

PHOSPHATE METABOLISM IN BLUE-GREEN ALGAE. II. CHANGES IN PHOSPHATE DISTRIBUTION DURING STARVATION AND THE “POLYPHOSPHATE OVERPLUS” PHENOMENON IN PLECTONEMA BORYANUM

Physiological aspects of phosphate utilization by the blue-green alga Plectonema boryanum were studied. It was found that the external phosphate concentration influenced the distribution of phosphorus-containing compounds in the cell. Culturing the alga in concentrations of 10, 100, and 1000 mg PO₄/l resulted in increases in the level of acid-soluble and acid-insoluble polyphosphates. The values reported for 100 and 1000 mg PO₄/l were the same, indicating that the cells were able to assimilate and utilize only fixed amounts of phosphates. The total phosphorus value for these cells was calculated to be 6.5 μg P per 10⁶ cells. Culturing the alga in 1 mg PO₄/l led to a decrease in phosphate concentration of all cell fractions. Cells grown in the absence of phosphate for 5 days had total cell phosphorus levels of 0.76 μg P per 10⁶ cells. Cells in culture for two months or longer were found to have total cell phosphorus levels of 0.73 μg P per 10⁶ cells. This was determined to be the minimum cell phosphorus level limiting growth. Transfer of cells from either culture condition to a medium containing phosphate led to an “overplus” phenomenon. This overplus phenomenon was characterized by increases in all cellular phosphorus fractions. The most dramatic increase was found in both the acid-soluble and acid-insoluble polyphosphates. These fractions often increased by more than an order of magnitude. The greatest phosphate uptake occurred within 1 hr of transfer of phosphate-starved cells into a medium containing a known amount of phosphate and is essentially complete at 4 hr. The total cell phosphorus levels for uptake never increased beyond 18.9 μg per 10⁶ cells.     

Inhibitor effects on photosynthesis,respiration and active ion transport inHydrodictyon africanum

Summary The effects of various inhibitors on photosynthesis, respiration, and active influx of K and Cl in light and dark inHydrodictyon africanum is reported. The inhibitors used were arsenate (uncouples electron-transport phosphorylations), dicyclohexylcarbodiimide (energy-transfer inhibitor in electron-transport phosphorylation), quinacrine (uncouples photophosphorylation and inhibits oxidative phosphorylation), and ethionine (traps adenylates as S-adenosyl ethionine). The action of these inhibitors, and of those previously used onHydrodictyon africanum, suggests that K influx requires ATP, while Cl influx requires some earlier manifestation of the ATP synthesizing process. Possible reasons for the greater sensitivity of K influx than of CO₂ fixation to treatments which interfere with photophosphorylation are discussed.     

Nitratabhängige nichtcyclische Photophosphorylierung bei Ankistrodesmus braunii in Abwesenheit von CO2 und O2

Summary Manometric measurements show that oxygen evolution proceeds in synchronised cells of Ankistrodesmus braunii even in an atmosphere of pure nitrogen. In this case the slow oxygen evolution is dependent on the presence of nitrate (Table 1). Light saturation is found at a low light intensity at pH 5.6, at a higher light intensity at pH 8.0 (Fig. 1). The light saturation curves are in good agreement with those of ³²P-labelling in Ankistrodesmus under the same conditions (Fig. 2).DCMU inhibition in N₂ of both O₂-evolution and ³²P-labelling begins only at a DCMU concentration of 5×10^-7M or more. Complete inhibition of O₂-evolution is reached only at 10^-5M (Fig.3). In ³²P-labelling a variable percentage is still left uninhibited at 10^-5 M DCMU (Fig. 4, Table 2), which is at least partly due to cyclic photophsphorylation. Nitrate starvation for several hours causes a considerable decrease in O₂-evolution and also in the sensitivity to those high concentrations of DCMU (Fig. 5), but it leads to a sensitivity to antimycin A not observed under normal conditions (Table 3). The effects of nitrate starvation thus become comparable to those of far-red light, under which noncyclic electron transport is slow or completely prevented.The inhibition by DCMU of electron transport in photosystem II is also estimated by measuring the increase in fluorescence at 684 nm in air containing additional CO₂. This fluorescence is saturated only at 10^-5M DCMU and shows that a certain percentage of photosystem II remains uninhibited at 5×10^-7M (Fig. 6), a concentration found to be almost ineffective in inhibiting O₂-evolution and ³²P-labelling in an N₂-atmosphere.The results indicate that in synchronised cells of Ankistrodesmus noncyclic electron flow and noncyclic photophosphorylation can proceed in an atmosphere of pure nitrogen if nitrate is available as the electron acceptor. In this case noncyclic photophosphorylation, inspite of its low rates, still dominates over cyclic photphosphorylation. At low pH, when nitrate reduction is slow, cyclic photophosphorylation accounts for a greater part of the total phosphorylation than at high pH. Thus in the absence of CO₂ and O₂ cyclic photophosphorylation can be regarded as the main process of ATP formation only after nitrate starvation, in far-red light or in the presence of high concentrations of DCMU.Inhibition by DCMU, though very efficient under conditions of high photosynthetic activity, becomes rate-limiting only if the electron transport is so far reduced by DCMU that the remaining rate is of the same order as the low rate of the control or less. Therefore high concentrations of DCMU are required for the inhibition of low rates of noncyclic photophosphorylation.     

Light-Induced Phosphate Binding in Relation to Photophosphorylation and Levels of ATP, ADP and AMP in the Green Alga Scenedesmus

Synchronous cultures of Scenedesmus obtusiusculus Chod. were starved for phosphorus for 48 h. Such cells develop an efficient mechanism for phosphate binding which is very sensitive to metabolic inhibitions. Phosphate binding, fluctuations in the ATP pool during dark-light-dark transitions, and steady state levels of ATP, ADP and AMP were studied. The experiments were carried out in a CO₂-free N₂ atmosphere. DCMU, phloridzin and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) were used as inhibitors of photophosphorylation. Light-induced phosphate uptake was inhibited to various extents by all the inhibitions. The dark-light-dark transition experiments show that neither the light-induced increment in ATP nor the decrease at darkening are affected by DCMU, but DBMIB and phloridzin inhibit both processes. DCMU seems to affect the regulation of the ATP pool size. The steady state levels of the adenylate pools were almost the same in the light as in the dark, and they were also little sensitive to the inhibitors. In unpoisoned cells in the light the steady state ATP/ADP ratio was 1.7 and the energy charge was 0.66. The rates of phosphate binding are not correlated to any of the adenylate parameters studied. This is probably due to the diverse effects of the inhibitors on light-stimulated production of reducing equivalents, photophosphorylation and transfer of energy from the chloroplast to the cytoplasm.     

The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 °C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit.2. CO₂ fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3. Phosphoglycerate reduction, which is a partial reaction of CO₂ fixation, shows very little pH dependency.3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO₂ fixation without affecting phosophoglycerate reduction. This inhibition of CO₂ fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP.4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO₂ fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO₂ fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport.5. It is shown that intact chloroplasts are able to fix CO₂ in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma.6. Dark CO₂ fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO₂ fixation in the light. This demonstrates that CO₂ fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.7. It is shown that pH changes occurring in the stroma after illumination are sufficient to switch CO₂ fixation from zero to maximal activity. This offers a mechanism for light control of CO₂ fixation, avoiding wasteful CO₂ fixation in the dark.     

Phosphorus incorporation in the ehrlich ascites tumor cell

Data from isotopic uptake experiments were used to measure the kinetics of labelling of cellular phosphate, ATP and ADP in the Ehrlich ascites tumor cell. The results show that steady state phosphate exchange flux was 0.333 ± 0.052 (S.E.) μmoles per 10⁷ cells per hour at 37°, and that the specific activity of phosphate was the same as P_γ ATP. Metabolic inhibition reduced the phosphate flux by 30–50%. A model, based on oxidative phosphorylation and the adenylate kinase reaction is used to interpret the labelling sequence of P_β ATP and P_β ADP, and its dependence on P_γ ATP.     

CO2-Fixation and ATP synthesis in continuous cultures of Chlorella fusca

In continuous cultures of Chlorella fusca under steady state conditions, the CO₂-fixation rate, the ATP-level, the apparent rate of photophosphorylation as calculated from the changes in the ATP-level during light to dark or dark to light transients and the energy charge were measured at various environmental conditions. During growth the energy charge was around 0.64. CO₂-assimilation and the apparent ATP-synthesis were strongly dependant on light intensity, however the ATP-level was independant on it. Since the rates of apparent ATP-synthesis and of the CO₂-fixation do not seem to be strictly correlated in a logic way when environmental factors are changed and furthermore the stoichiometry of 3 ATP necessary per CO₂ fixed was never achieved, the described method frequently used for procaryotes to determine the in vivo rate of phosphorylation does not give valid results in highly compartimented eukaryotic cells.     

Physiological performances of the blue-green alga Anacystis nidulans in continuous turbidostat fermenter culture

A sterile continuous turbidostat culture in a 2-1 fermenter was used to systematically measure the gas exchange rates of Anacystis nidulans in a highly turbulent system under strictly controlled environmental conditions. An extensive physiological characterization of Anacystis is given in terms of photosynthesis rates (CO₂ uptake and O₂ evolution) and dark respiration rates as function of different parameters such as stirrer speed, temperature, CO₂ and O₂ concentration, light intensity, culture density and pH. Steady state ATP levels and apparent photophosphorylation rates complete the performance data. The dependence of the photosynthetic quotient from the parameters enables a physiological characterization of the light dependent nitrate assimilation.     

Die Wirkung von Dinactin auf die cyclische Photophosphorylierung,die lichtinduzierte ATP-Pa-Austauschreaktion und den lichtinduzierten Protonentransport in Chloroplasten

Summary Dinactin, an antibiotic forming complexes with K⁺ ions, uncouples phosphorylation in chloroplasts without requiring the presence of a substance increasing the permeability of the membrane for protons. To inhibit photophosphorylation, less Dinactin is necessary in the absence than in the presence of K⁺.When added before the light phase, Dinactin affects the light-triggered ATP-P_i exchange reaction in the same way as it does the complete photophosphorylation. Addition of the antibiotic after the activation by light inhibits the exchange reaction independently of the presence of K⁺, possibly by blocking the energy transfer to ATP.The inhibition of the light-induced proton transport by Dinactin is more pronounced in the presence of K⁺ than of Na⁺ ions. The manner in which changes in the permeability of the chloroplast membrane for K⁺ ions caused by Dinactin may influence photophosphorylation and reactions coupled with it is discussed.

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Verwendete Abkürzungen ATP Adenosintriphosphat - ADP Adenosindiphosphat - P_a anorganisches Phosphat - PMS Phenazinmethosulfat - DCPIP Dichlorphenolindophenol - FeCy Ferricyanid - DNP Dinitrophenol - FCCP Carbonylcyanid-p-trifluormethoxyphenylhydrazon - SQ 15859 Squibb Compound 15859     

Inhibition of photophosphorylation by ATP and the role of magnesium in photophosphorylation

ATP and pyrophosphate at high concentration (> 1 mM) inhibited photophosphorylation of isolated spinach chloroplasts in the normal salt medium and did not cause stimulation of electron transport. The inhibition of photophosphorylation by ATP or pyrophosphate was shown to be abolished by the addition of excess MgCl₂, ADP and phosphate. It has been demonstrated that the rates of photophosphorylation in the absence and presence of ATP or pyrophosphate are determined similarly by the concentrations of magnesium-ADP (Mg · ADP^?) and magnesium-phosphate (Mg · P_i) complexes.It is highly probable that Mg · ADP^? and Mg · P_i, but not free ADP and free phosphate, are the active form of the substrates of photophosphorylation. This is in support of the view that ATP inhibits photophosphorylation by decreasing the concentration of Mg²⁺ which is available for the formation of the complex with ADP and phosphate.     

Der Einfluß von Natrium- und Kalium-Ionen auf die Photophosphorylierung bei Ankistrodesmus braunii

Summary During short-time experiments (30 sec to 60 min) sodium ions stimulate the phosphate uptake and especially the ³²P-labelling of the organic TCA-soluble phosphate compounds up to 1,500% (K⁺=100%). The labelling is maximally stimulated in the light and in the dark at concentrations of about 5×10^-3 mol/l Na⁺ and at pH 8. Lithium ions stimulate ³²P-labelling in a similar but less effective way. In comparison, in the presence of potassium ions the ³²P-label decreases.It was investigated whether sodium ions specifically stimulate the ATP-synthesis or some reaction of the photosynthetic carbon reduction cycle or whether they only enhance the ³²P-labelling of phosphorylated compounds.Separation by thin-layer chromatography of the MCF-soluble phosphate fraction showed that labelling of all compounds investigated was stimulated by Na⁺ to a similar extent.Experiments performed in red and far-red light (683 and 712 nm) under nitrogen and in the presence of various DCMU-concentrations, as well as in the presence of antimycin A and CCCP showed that Na⁺ exerts no specific influence either on the cyclic or on the non-cyclic photophosphorylation in vivo.ATP-dependent reactions such as ¹⁴CO₂-fixation or glucose uptake are not influenced by Na⁺.Since Na⁺ does not change the size of phosphate pools in a different way from K⁺, there is no evidence for the assumption that the Na⁺-dependent increase in the ³²P-labelling is due to its action on the chloroplast membrane in increasing its permeability to orthophosphate ions. This is supported by the lack of any effect of sodium plus phosphate ions on the CO₂-fixation.Therefore the results give no evidence that sodium acts directly on phosphorus metabolism inside the cell. It is suggested that its action is localised at the phosphate-transporting site of the plasmalemma.     

Control of electron flow in intact chloroplasts by the intrathylakoid pH,not by the phosphorylation potential

In the presence of nitrite or oxaloacetate, intact chloroplasts evolved oxygen at a significant rate for the initial 1 to 2 min of illumination. Subsequently, oxygen evolution was suppressed progressively. The suppressed oxygen evolution was stimulated strikingly by NH₄Cl. The results indicate that coupled electron flow in intact chloroplasts is controlled in the light, and the control is released by NH₄Cl. However, at low concentrations, NH₄Cl was not an effective uncoupler of photophosphorylation in intact chloroplasts. Intrachloroplast ATP levels and ATP/ADP ratios were not significantly influenced by NH₄Cl. In contrast, the quenching of 9-aminoacridine fluorescence, which can be used to indicate the intrathylakoid pH in intact chloroplasts, was reduced drastically even by low concentrations of NH₄Cl. This suggests that the chloroplast phosphorylation potential is not in equilibrium with the proton gradient. In coupled chloroplasts, the intrathylakoid pH was lower in the light with nitrite than with oxaloacetate as electron acceptor. Electron flow was also more effectively controlled in chloroplasts illuminated with nitrite than with oxaloacetate. It is concluded that the intrathylakoid pH, not the phosphorylation potential, is a factor in the control of the rate of electron flow in intact chloroplasts.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - OAA oxalo-acetate - MES 2-(N-morpholino)-ethanesulfonic acid - HEPES N-2-hyroxyethylpiperazine-N-2-ethanesulfonic acid Postal address     

Regulation of photosynthetic electron transport and photophosphorylation in intact chloroplasts and leaves of Spinacia oleracea L.

Oxygen ist reduced by the electron transport chain of chloroplasts during CO₂ reduction. The rate of electron flow to oxygen is low. Since antimycin A inhibited CO₂-dependent oxygen evolution, it is concluded that cyclic photophosphorylation contributes ATP to photosynthesis in chloroplasts which cannot satisfy the ATP requirement of CO₂ reduction by electron flow to NADP and to oxygen. Inhibition of photosynthesis by antimycin A was more significant at high than at low light intensities suggesting that cyclic photophosphorylation contributes to photosynthesis particularly at high intensities. Cyclic electron flow in intact chloroplasts is under the control of electron acceptors. At low light intensities or under far-red illumination it is decreased by substrates which accept electrons from photosystem I such as oxaloacetate, nitrite or oxygen. Obviously, the cyclic electron transport pathway is sensitive to electron drainage. In the absence of electron acceptors, cyclic electron flow is supported by far-red illumination and inhibited by red light. The inhibition by light exciting photosystem II demonstrated that the cyclic electron transport pathway is accessible to electrons from photosystem II. Inhibition can be relieved by oxygen which appears to prevent over-reduction of electron carriers of the cyclic pathway and thus has an important regulatory function. The data show that cyclic electron transport is under delicate redox control. Inhibition is caused both by excessive oxidation and by over-reduction of electron carriers of the pathway.     

Operation of the glycolate pathway in isolated bundle sheath strands of maize and Panicum maximum

Operation of the glycolate pathway in isolated bundle sheath (BS) strands of two C₄ species was demonstrated from ¹⁴C incorporation into two intermediates, glycine and serine, under conditions favourable for photorespiratory activity. Isolated BS strands fixing ¹⁴CO₂ under light at physiological rates incorporate respectively 3% (Zea mays L., cv. INRA 258) and 7% (Panicum maximum Jacq.) of total ¹⁴C fixed into glycine + serine, at low bicarbonate levels (less than the Km for CO₂ fixation, 0.8 mM). Higher bicarbonate concentrations depressed the percentage of incorporation into the two amino acids. No labelling was observed in the absence of added glutamate. Oxygen was required for glycine + serine labelling, since ¹⁴C incorporation into glycine was largely depressed by argon flushing, and labelling of the two amino acids was nearly suppressed by the addition of the strong reductant, dithionite, especially in maize. Two inhibitors of the glycolate pathway were tested. With α-hydroxypyridine-methanesulfonic acid, an inhibitor of glycolate oxidase, labelling of glycine and serine remained minimal whereas glycolate was accumulated. Isoniazid, an inhibitor of the transformation of glycine to serine induced a 50% increased labelling of glycine in maize BS, and a large decrease in serine labelling. In Panicum, the increase in [¹⁴C]-glycine was 90%. These results suggest that the pathway glycolate → glycine → serine operates in these plants. However, leakage of metabolites occurs in BS cells, especially in maize and a large part of newly formed glycolate, glycine and serine is exported out of the cells. Operation of ribulose-1,5-bisphosphate oxygenase activity in competition with ribulose-1,5-bisphosphate carboxylase is demonstrated by the lowering of total ¹⁴CO₂ fixation when O₂ is increased at low bicarbonate concentration. An interesting feature observed in maize BS, at low bicarbonate concentration, was an increase in ribulose-1,5-bisphosphate labelling when the O₂ level was decreased. This was accompanied by an increase in CO₂ fixation. This could indicate an increased rate in synthesis of ribulose-1,5-bisphosphate (which accumulated) due to a stimulation of ATP synthesis by cyclic photophosphorylation under anaerobic conditions.     

Energy-dependent changes in the ATP/ADP ratio at the tight nucleotide binding site of chloroplast ATP synthase

Using DTT-modulated thylakoid membranes we studied tight nucleotide binding and ATP content in bound nucleotides and in the reaction mixture during [¹⁴C] ADP photophosphorylation. The increasing light intensity caused an increase in the rate of [¹⁴C] ADP incorporation and a decrease in the steady-state level of tightly bound nucleotides. Within the light intensity range from 11 to 710 w m^–2, ATP content in bound nucleotides was larger than that in nucleotides of the reaction mixture; the most prominent difference was observed at low degrees of ADP phosphorylation. The increasing light intensity was accompanied by a significant increase of the relative ATP content in tightly bound nucleotides. The ratio between substrates and products formed at the tight nucleotide binding site during photophosphorylation was suggested to depend on the light-induced proton gradient across the thylakoid membrane.Abbreviations AdN adenine nucleotide - Chl chlorophyll - DTT dithiothreitol - FCCP carbonylcianide p-trifluoromethoxyphenilhydrazone - P_i inorganic orthophosphate - PMS phenazine methosulfate - TLC thin-layer chromatography - Tricine N-[tris(hydroxymethyl)methyl] glycine