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.     

Der Einfluß von Natrium- und Kaliumionen auf die Phosphataufnahme bei Ankistrodesmus braunii

Summary The uptake of phosphate as influenced by sodium and potassium ions was investigated in the light and in the dark. It was found to be a function of the external phosphate concentration. At a low concentration (up to 10^–5 mol/l) in the presence of Na⁺ phosphate is quickly absorbed and hence phosphate is the limiting factor for further labelling. In the presence of K⁺ phosphate uptake is constant over a long period.The enhancement of phosphate uptake by Na⁺ is also found when the external concentration of P is raised up to 10^–4 mol/l. Then the gross uptake proceeds over six hours, with the greatest Na⁺-dependent increase occurring in the label of the TCA-insoluble phosphate fraction (P_u).The phosphate uptake is strongly dependent on the pH of the reaction mixture. In the presence of Na⁺ it is highest between pH 5.6 and 7. As the uptake in the presence of K⁺ parallels the dissociation curve of the dihydrogen form H₂PO ₄ ^– , the Na⁺-enhancement is optimal in the alkaline pH range (pH 8).On the basis of a comparison between the pH-dependence of phosphate uptake and the dependence of the uptake on the external phosphate concentration analysed by a method of enzyme kinetics, it is suggested that Ankistrodesmus metabolically transports H₂PO ₄ ^– but not HPO ₄ ⁼ . Moreover, it is concluded from the absence of light stimulation and the weak inhibition of the uptake by DCMU or CCCP in the presence of K⁺ that at low P-concentrations the diffusion is limiting the uptake. Only at higher concentrations is an active phosphate uptake measured.Furthermore it is concluded that the observed Na⁺-stimulation of the ³²P-labelling of the TCA-soluble and insoluble compounds inside the cell is indirect and depends only on the action of Na⁺ and K⁺ ions at the first transport site in the plasmalemma.     

Das Verhalten von Nitratreductase,Nitritreductase, Hydrogenase und anderen Enzymen von Ankistrodesmus braunii bei Stickstoffmangel

Zusammenfassung Bei der N-Verarmung von Ankistrodesmus braunii wurde eine intracelluläre Bildung von Nitrit und Nitrat durch heterotrophe Nitrifikation festgestellt, die charakteristisch für den beginnenden N-Mangel ist. Damit wird die hohe Nitrat- und Nitritreductaseaktivität bei N-Mangelalgen verständlich.Die Hydrogenase ist auch bei N-verarmten Algen aktiv, doch kann Nitrit nicht mehr als H-Acceptor verwendet werden. Die Aktivierung des Enzyms ist energieabhängig und durch Antibiotica (Actinomycin C, Puromycin, Gentamycin) hemmbar. Offenbar findet während der Inkubation mit Wasserstoff eine Proteinsynthese statt.Zum Vergleich wurde das Verhalten einiger anderer Enzyme [Glucose-6-phosphat-dehydrogenase, NAD(P)-Reductase, Glyoxylat-reductase, Katalase, Malat-dehydrogenase, Glutamat-dehydrogenase, Isocitratase] bei N-Mangel untersucht.

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Nitrate reductase, nitrite reductase, hydrogenase, and other enzymes in nitrogen-deficient Ankistrodesmus braunii

Summary An oxidation of organic nitrogen compounds leading to an intracellular formation of nitrite and nitrate (heterotrophic nitrification) was found in nitrogen-deficient Ankistrodesmus braunii. This explains the rather high levels of nitrate and nitrite reductases observed in algae after the supply of nitrogen has been exhausted.Hydrogenase is active also in nitrogen-deficient algae which, however, can no longer use nitrite as an acceptor for hydrogen. The activation of hydrogenase is energy-dependent and can be inhibited by means of antibiotics (actinomycin C, puromycin, and gentamycin). Protein synthesis seems to take place during incubation under hydrogen.For comparison, several other enzymes [glucose-6-phosphate dehydrogenase, NAD(P) reductase, glyoxylate reductase, catalase, malate dehydrogenase, glutamate dehydrogenase, and isocitratase) were studied in nitrogen-deficient cells.

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Verwendete Abkürzungen G-6-P-DH Glucose-6-phosphat-dehydrogenase - M-DH Malat-dehydrogenase - Glu-DH Glutamat-dehydrogenase - NAD(P)-R Nicotinamid-adenin-dinucleotid(phosphat)-reductase - CCCP Carbonylcyanid-mchlorophenylhydrazon - DNP 2,4-Dinitrophenol - MB Methylenblau     

Beziehungen der Aufnahme von Nitrat,Nitrit und Phosphat zur photosynthetischen Reduktion von Nitrat und Nitrit und zum ATP-Spiegel bei Ankistrodesmus braunii

Summary The pH-dependence of NO ₃ ^- and NO ₂ ^- uptake is different from that of phosphate uptake, but similar to that of sulfate uptake, with optima between pH 7.4 and 8.2 and smaller peaks at higher H⁺-concentration.Since the ATP-level is not affected by addition of ions and since phosphate uptake is not depressed by NO ₃ ^- , the inhibition of phosphate uptake by K⁺ reported in former papers cannot be explained by competition for the available energy(ATP) at the site of uptake.NO ₃ ^- uptake is strongly dependent on the activity of the NO ₃ ^- reducting system, as can be seen from the inhibition of NO ₃ ^- uptake in light by N₂ compared with that in air. Furthermore, the pH-dependences of NO ₃ ^- and NO ₂ ^- uptake correspond to the pH-optima known for the reductases.Phosphate uptake is enhanced by NO ₃ ^- and NO ₂ ^- in N₂. Since the enhancement of phosphate uptake is sensitive to DCMU and since this DCMU-sensitive phosphate uptake is accompanied by O₂-evolution, it is probably due to an NO ₃ ^- -stimulated noncyclic photophosphorylation which enhances the ATP-turnover and hence the incorporation of phosphate into organic compounds.

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Abkürzungen TCE Trichloressigsäure - P Orthophosphat - P₀ TCE-lösliche organische Phosphatverbindungen - P_u TCE-unlösliche Phosphatverbindungen - GP Gesamtphosphat     

Zur Wirkung von Sauerstoff auf die 32P-Markierung von Polyphosphaten und organischen Phosphaten bei Ankistrodesmus braunii im Licht

Summary Short time incorporation of ³²P was carried out with synchronised algae (young cells) depleted of phosphate. For the separation and determination of the acid-insoluble phosphate fractions of the cells an improved fractionation procedure was applied. In order to exclude competition by carbon dioxide all experiments were done in the absence of CO₂.Compared with nitrogen, CO₂-free air produces an increase in the labelling of phosphorylated compounds in the light. In strong white light, at high pH, air effects a remarkable increase of ³²P in the acid-insoluble phosphate (P _u), mainly in inorganic polyphosphates (P _ul), whereas the total phosphate uptake remains almost unchanged. The increase in labelling of acid-insoluble phosphate is, therefore, accompanied by a substantial decrease in the labelling of acid-soluble compounds (P _l). In weak white light or in far-red light, at low pH even in strong white or red light, an increase of phosphate uptake and an increased labelling of the acid-stable organic acid-soluble fraction (P _os) is observed instead. The effect of oxygen increases somewhat with increasing light intensity up to light saturation, and it increases markedly with increasing oxygen concentration.An essential contribution by oxidative phosphorylation to this oxygen effect can be ruled out on account of its much higher sensitivity to oxygen. Pseudocyclic photophosphorylation is also not regarded as the main force because of its higher oxygen affinity. Occurrence of photorespiration has not been clearly established so far in related algae (Chlorella), and its use for phosphorylation is unknown. A better, although not complete explanation is given by comparing the oxygen effect with the well-known inhibition of photosynthesis by oxygen (Warburg effect), which leads to an increase in glycolate formation and a simultaneous decrease in the pool sizes of carbon reduction cycle intermediates, even in the absence of CO₂. Since the photophosphorylation process, as well as the photosynthetic electron flow, seem unaffected by high oxygen concentrations whereas the formation of organic phosphate compounds is partially inhibited, excess ATP may be available for polyphosphate synthesis. This explanation would be consistent with the assumption that polyphosphate-ADP kinase mediates an equilibrium between ATP and polyphosphates, mainly at higher pH. At low pH and in other cases the excess ATP might be available for an increased phosphate uptake and for phosphorylation of endogenous carbohydrates.

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Herrn Prof. Dr. W. Simonis zum 60. 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.     

Studies on the regulation of assimilatory nitrate reductase in Ankistrodesmus braunii

In the green alga Ankistrodesmus braunii, all the activities associated with the nitrate reductase complex (i.e., NAD(P)H-nitrate reductase, NAD(P)H-cytochrome c reductase and FMNH₂-or MVH-nitrate reductase) are nutritionally repressed by ammonia or methylamine. Besides, ammonia or methylamine promote in vivo the reversible inactivation of nitrate reductase, but not of NAD(P)H-cytochrome c reductase. Subsequent removal of the inactivating agent from the medium causes reactivation of the inactive enzyme. Menadione has a striking stimulation on the in vivo reactivation of the inactive enzyme. The nitrate reductase activities, but not the diaphorase activity, can be inactivated in vitro by preincubating a partially purified enzyme preparation with NADH or NADPH. ADP, in the presence of Mg²⁺, presents a cooperative effect with NADH in the in vitro inactivation of nitrate reductase. This effect appears to be maximum at a concentration of ADP equimolecular with that of NADH.Abbreviations ADP Adenosine-5-diphosphate - AMP Adenosine-5-monophosphate - ATP Adenosine-5-triphosphate - FAD Flavin adenine dinucleotide - FMNH₂ Flavin adenine mononucleotide, reduced form - GDP Guanosine-5-diphosphate - MVH Methyl viologen, reduced form - NADH Nicotinamide adenine dinucleotide, reduced form - NADPH Nicotinamide adenine dinucleotide phosphate, reduced form     

Die pH-Abhängigkeit der Aufnahme von H2PO 4 - , SO 4 = , Na+ und K+ und ihre gegenseitige Beeinflussung bei Ankistrodesmus braunii

Summary Ion uptake was studied using ³²P, ³⁵S, ²²Na and ⁴²K as tracers in synchronized cells of Ankistrodesmus, which were slightly starved with respect to the ions to be investigated. In the light and in the dark, phosphate uptake is maximal between pH 5.5 and 6.5. Whereas Na⁺ in comparison to K⁺ enhances phosphate uptake in the light (8 to 9-fold) and in the dark, Ca⁺⁺ exerts only a slightly stimulatory effect. The stimulation of phosphate binding by Na⁺ occurs rapidly, even after less than 5 sec of incubation, and also in the presence of an equimolar concentration of K⁺.The pH-dependence of Na⁺-uptake in the light and in the dark is comparable to a dissociation curve: Na⁺-uptake increases with decreasing extracellular H⁺-concentration and is inversely proportional to phosphate uptake in the absence of Na⁺. The light:dark ratio of Na⁺-uptake at pH 8 amounts to 7:1. Mere adsorption of Na⁺ is similarly dependent on the pH. K⁺ strongly competes with Na⁺-uptake, even at pH 8. K⁺-uptake proceeds in a quite different manner from Na⁺-uptake and has an optimum at pH 7.Sulfate is taken up linearly in a biphasic process as a function of time; the pH-optimum lies between pH 7.5 and 8. K⁺ but not Na⁺ slightly enhances sulfate uptake.The Na⁺-enhancement of phosphate uptake can be related neither to a sodium-potassium exchange pump nor to a photosynthesis-dependent ion-exchange reaction.The results suggest that the uptake of phosphate, Na⁺ and K⁺, and the influence of alkali cations on phosphate uptake, but not sulfate uptake, are strongly dependent on fixed charges of the plasmalemma or even of the cell wall. These fixed charges may even prevent an active ion uptake.     

Intracellular appearance of nitrite and nitrate in nitrogen-starved cells of Ankistrodesmus braunii

Summary The occurrence of heterotrophic nitrification in nitrogen-starved cells of Ankistrodesmus braunii was confirmed. The levels of nitrate and nitrite were measured over a period of four weeks. The validity of quantitative determinations in the presence of highly active nitrate and nitrite reductases is discussed. Whereas free hydroxylamine as an intermediate could not be detected, increased hydroxylamine oxidase activity was found in nitrogen-starved cultures. Nitrite reductase and hydroxylamine oxidase can be assigned to particles by sucrose density gradient centrifugation. The possible involvement of microbodies, which were found to be present in Ankistrodesmus, in metabolic processes during nitrogen starvation is discussed.Abbreviations NR nitrate reductase - NiR nitrite reductase - NNEDA N-(1-naphthyl)ethylenediaminedihydrochloride - DCPIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - TCA trichloroacetic acid - DAB 3,3-diaminobenzidine - AT 3-amino-1H-1,2,4-triazole - AMP 2-amino-2-methyl-1,3-propanediol     

The appearance of nitrate reductase activity in nitrogen-starved cells of Ankistrodesmus braunii

Summary Crown-gall teratoma tissues of tobacco, when grown in culture, require exogeneous auxin (-naphthaleneacetic acid) or high concentrations of K⁺ in the medium to utilize ammonium glutamate as a nitrogen source. These factors are not required to utilize NO ₃ ^- or glutamine. The effects of K⁺ and auxin on glutamate utilization differ in that NH ₄ ⁺ is required for the action of K⁺, but not for the action of auxin. The tissues grew optimally when the ratio of NH ₄ ⁺ to glutamate was approximately one or greater. These results indicate that glutamate utilization involves at least two different mechanisms: one mechanism requires K⁺ and stoichiometric amounts of NH ₄ ⁺ , the other mechanism requires auxin. Experiments using explants of tobacco pith show that both mechanisms function in normal as well as crown-gall tissues of tobacco.     

Characterization of the Reversible Inactivation of Ankistrodesmus braunii Nitrate Reductase by Hydroxylamine

The photoreversible nature of the regulation of nitrate reductase is one of the most interesting features of this enzyme. As well as other chemicals, NH₂OH reversibly inactivates the reduced form of nitrate reductase from Ankistrodesmus braunii. From the partial activities of the enzyme, only terminal nitrate reductase is affected by NH₂OH. To demonstrate that the terminal activity was readily inactivted by NH₂OH, the necessary reductants of the terminal part of the enzyme had to be cleared of dithionite since this compound reacts chemically with NH₂OH. Photoreduced flavins and electrochemically reduced methyl viologen sustain very effective inactivation of terminal nitrate reductase activity, even if the enzyme was previously deprived of its NADH-dehydrogenase activity. The early inhibition of nitrate reductase by NH₂OH appears to be competitive versus NO₃⁻. Since NO₃⁻, as well as cyanate, carbamyl phosphate and azide (competitive inhibitors of nitrate reductase versus NO₃⁻), protect the enzyme from NH₂OH inactivation, it is suggested that NH₂OH binds to the nitrate active site. The NH₂OH-inactivated enzyme was photoreactivated in the presence of flavins, although slower than when the enzyme was previously inactivated with CN⁻. NH₂OH and NADH concentrations required for full inactivation of nitrate reductase appear to be low enough to potentially consider this inactivation process of physiological significance.     

Biomass production from the green algae Chlorella vulgaris and Ankistrodesmus braunii cultured heterotrophically

Summary Ankistrodesmus braunii and Chlorella vulgaris were cultured heterotrophically under various operating conditions. The maximum rate of biomass production was 900 and 900 mg L^-1 d^-1 by C. vulgaris and 1000 and 700 mg L^-1 d^-1 by A. braunii in the light and dark, respectively. This indicates that these algae could produce in excess of 1530 dry weight tonnes ha^-1 y^-1 which is 10–20 times higher than the maximum production levels in the literature.     

Affinity chromatography of Ankistrodesmus braunii nitrate reductase using blue dextran-sepharose (author's transl)

Blue Dextran has been coupled covalently to Sepharose-4B to purify the enzymatic complex NAD(P)H-nitrate reductase (EC 1.6.6.2) from the green alga Ankistrodesmus braunii by affinity chromatography. The optimum conditions for the accomplishment of the chromatographic process have been determined. The adsorption of nitrate reductase on Blue Dextran Sepharose is optimum when a phosphate buffer of low ionic strength and pH 6.5-7.0 is used. Once the enzyme has been bound to Blue Dextran Sepharose, it can be specifically eluted by addition of NADH and FAD to the washing buffer. However, none of the nucleotides added separately is able to promote the elution of the enzyme from the column. The elution can be also achieved, but not specifically, by increasing the ionic strength of the buffer with KCl. These results have made possible a procedure for the purification of A. braunii nitrate reductase which led to electrophoretic homogeneity, with an overall yield of 70% and a specific activity of 49 units/mg of protein.     

Hohe Strahlenresistenz der Aktivität und der lichtinduzierten Aktivitätssteigerung der NADP-abhängigen Glycerinaldehyd-3-Phosphat-Dehydrogenase in Ankistrodesmus braunii

Summary In Ankistrodesmus braunii, a unicellular alga, the activity of NADP-dependent glyceraldehyde phosphate dehydrogenase (GPD) in darkness is very resistant to X-rays. The light-induced increase in the activity of this enzyme within one minute is also not affected by irradiation. Doses up to 1060 Krad had no inhibitory effect. The increase of GPD activity during the life cycle, however, is very sensitive to X-rays.