Light Stimulation of Active Transport in Hydrodictyon africanum

The mechanism of light stimulation of active K and Cl influx and active Na efflux, in Hydrodictyon africanum has been investigated using different wavelengths of red light and different gas mixtures, and the inhibitors DCMU and CCCP. The active Cl influx requires photosystem 2, since its relative quantal efficiency falls with increasing wavelength of red light, and it is as sensitive to the inhibitor DCMU as is photosynthesis; it is relatively insensitive to the uncoupler CCCP. The active K influx and active Na efflux are inhibited by CCCP, but the relative quantal efficiency of these processes increases with increasing wavelength of red light, and they are relatively insensitive to DCMU. These cation fluxes can be supported by cyclic photophosphorylation, whereas Cl influx needs photosystem 2 but probably not ATP.     

The Effect of Dio-9 on Photosynthesis and Ion Transport in Nitella, Tolypella, and Hydrodictyon

The effect of Dio-9, on photosynthesis and active and passiveion transport at the plasmalemma has been investigated in Nitellatranslucens, Tolypella intricata, and Hydrodictyon africanum.The active K influx and the coupled active Na efflux were moreinhibited by this inhibitor of energy transfer in phosphorylationthan was photosynthesis. The active Cl influx, the associateddownhill cation influxes, and passive ion fluxes were inhibitedby Dio-9, to a smaller extent than was photosynthesis. Cl influxin the light was often stimulated at concentrations of Dio-9,which inhibited photosynthesis. It was concluded that the activeK influx and Na efflux require ATP, while the active Cl influxdoes not. Possible links between the Cl influx and electrontransport and intermediates of photophosphorylation, and possibleinhibition by Dio-9 of the transport ATPase in the plasmalemmaare discussed.     

Cyclic and Non-cyclic Photophosphorylation as Energy Sources for Active K Influx in Hydrodictyon africanum

Under anaerobic conditions in the light, active K influx inHydrodictyon africanum is supported by cyclic photophosphorylation.The use of selective inhibitors shows that, in the presenceof CO₂, a considerable portion of the ATP used by the K pumpis supplied by noncyclic photophosphorylation. The rest of theATP in these conditions comes from cyclic photophosphorylation.This is true under light-limiting as well as light-saturatedconditions. If non-cyclic photophosphorylation is inhibited (by removalof carbon dioxide, by the addition of cyanide which interfereswith the carboxylation reaction, or by inhibition of photosystemtwo with DCMU or supplying only far-red light), the K influxat low light intensities is stimulated, and its characteristicsbecome those of a process powered by cyclic photophosphorylationalone. These results are interpreted in terms of a competitionfor ATP between K influx and CO₂ fixation. Implicit in thisexplanation is a requirement for a switch of excitation energyabsorbed by photosystem one from cyclic photophosphorylationto non-cyclic photophosphorylation whenever conditions (presenceof CO₂and photosystem two activity) allow CO₂ fixation to occur. Further evidence for such a switch of excitation energy absorbedby photosystem one was obtained in experiments in which redand far-red light were applied separately and together. It wasfound that CO₂ fixation showed the Emerson enhancement effect,while K influx (in the presence of CO₂) shows a ‘de-enhancement’.This suggests that far-red light alone powers cyclic photophosphorylation;if red light is also present, some of the far-red quanta arediverted to non-cyclic photophosphorylation. The nature of the interaction between cyclic and non-cyclicphotophosphorylation is discussed in relation to these and otherpublished results.     

The Action of Phlorizin on Photosynthesis and Light-stimulated Ion Transport in Hydrodictyon africanum

Phlorizin at 1 mM inhibits the coupled active influx of K andefflux of Na in Hydrodictyon africanum in the light. It doesnot inhibit the coupled influx of Cl and monovalent cations,nor the passive ion fluxes. Photosynthesis can be stimulatedup to 30 per cent under light-saturated conditions. It is concludedthat the effects of phlorizin cannot be solely due to an inhibitionof the membrane ATPase, and that photophosphorylation must insome way be affected. The nature of this effect is discussedin the light of the effects of phlorizin on photophosphorylationin isolated chloroplasts.     

Energetics of Active Phosphate Influx in Hydrodictyon africanum

The energy source for active phosphate influx in Hydrodictyonafricanum has been investigated using gas mixtures with andwithout O₂ and CO₂, light of various wavelengths, and metabolicinhibitors selective for respiratory or photosynthetic electrontransport and phosphorylation. It is concluded that, as in theother green algae studied, active phosphate transport requiresATP. In the dark this is supplied by oxidative phosphorylation;in the light the influx is much less sensitive to inhibitionof oxidative phosphorylation, and photophosphorylation (includingcyclic photophosphorylation) can act as energy source. Thissituation is more like that for active K influx (coupled toactive Na efflux) than to active Cl influx in H. africanum,except that the active dark influx is relatively greater forphosphate influx. The significance of these results for themechanism of regulation of light-stimulated ATP-requiring processes,and for the role of photosynthetic and oxidative phosphorylationin the energy metabolism of green cells, is discussed.     

Ouabain-insensitive K influx in Hydrodictyon africanum

Summary The occurrence is reported of cells of Hydrodictyon africanum which have, contrary to previous reports by the author, a K influx which is almost insensitive to ouabain. The conditions which govern the ouabain-sensitivity of the K influx have not yet been defined. The low ouabain sensitivity of the total K influx seems to be related to a smaller than usual activity of the component of K influx which is linked to Na efflux, and also to a smaller sensitivity of this component to inhibition by ouabain. The major components of K influx in ouabain-insensitive cells correspond to those components previously described as the passive and Cl-linked components.The occurrence is also reported of an increased sensitivity of active Cl influx (and the coupled cation influxes) in the light to uncouplers when the medium is aerated or otherwise stirred.     

The Linkage of Light-stimulated Cl Influx to K and Na Influxes in Hydrodictyon africanum

There are reciprocal stimulations of Cl influx by K and Na,and of K and Na influx by Cl, in the light in Hydrodictyon africanum.The component of the K influx which stimulates, and is stimulatedby, Cl, is independent of the ouabainsensitive mechanism forK influx also found in H. africanum. The concentration dependenceof the cation effects on Cl influx and on the Cl-stimulatedportion of their own influxes are similar. The stimulation withK saturates at about 0.3 mM K; that with Na saturates at about2 mM Na. The Cl-dependent portions of the K and Na influxeshave similar responses to changes in photo-synthetic metabolism(far-red illumination, CDMU, and CCCP) as does the light-stimulatedCl influx. This suggests that Cl influx, and the Cl-stimulatedportions of K and Na influxes are both dependent on photosystem2 of photosynthesis, and are less sensitive to the uncouplerCCCP than is ¹⁴CO₂ fixation or the K-Na pump. It would thusappear that the Cl-dependent portions of the K and Na influxesin the light are linked to the cation-stimulated portion ofthe Cl influx. There is no very great change in the electricalcomponent of the inwardly directed passive driving force oncations under conditions in which Cl is being pumped comparedwith those under which it is not. It is not clear whether suchincrease in this driving force as do occur could account quantitativelyfor the increase in the cation influxes associated with Cl transport,or whether chemical coupling must be invoked. In addition tothe Cl-stimulated portions of the cation influxes, there arealso light-stimulated portions of K and Na influx which areindependent of Cl, not associated with the cation regulatingmechanism, and which seem to have a similar linkage to photosynthesisas does the Cl-K-Na pump. Since the light-stimulated portionof the K efflux appears to be similar to this portion of theK influx, these Cl-independent light-stimulated portions ofK and Na influxes are tentatively related to light-induced changesin cation permeability.     

Ion Transport in Hydrodictyon africanum

The concentrations of K, Na, and Cl in the cytoplasm and vacuole, the tracer fluxes of these ions into and out of the cenocyte, and the electrical potential difference between bathing solution and vacuole and cytoplasm, have been measured in Hydrodictyon africanum. If the ions were acted on solely by passive electrochemical forces, a net efflux of K and Cl and a net influx of Na would be expected. Tracer fluxes indicate a net influx of K and Cl and efflux of Na in the light; these net fluxes are consequently active, with an obligate link to metabolism. The effects of darkness and low temperature indicate that most of the tracer K and Cl influx and Na efflux are linked to metabolism, while the corresponding tracer fluxes in the direction of the free energy gradient are not. Ouabain specifically inhibits the metabolically linked portions of tracer K influx and Na efflux. Alterations in the external K concentration have similar effects on metabolically mediated K influx and Na efflux. It would appear that K influx and Na efflux are linked, at least in the light.     

The Role of Cyclic and Pseudocyclic Photophosphorylation in Photosynthetic 14CO2 Fixation in Hydrodictyon africanum

Experiments are reported in which the effects on photosynthesisof various inhibitors of cyclic photophosphorylation were investigated.These inhibitors, generally had only a small inhibitory effecton photosynthesis, and the inhibition was not increased by conditionswhich inhibit pseudocyclic photophosphorylation. These inhibitorsdo not inhibit the Emerson enhancement effect. From these resultsit was concluded that photosynthesis does not need any ATP otherthan that produced in non-cyclic photophosphorylation. The effectsof these inhibitors on active K influx in light-anaerobic conditionsin the presence or absence of CO₂ suggest that some of the ATPproduced by non-cyclic photophosphorylation can be used to supportactive K influx. The results are discussed in relation to themechanism of the Emerson effect, the stoichiometry of non-cyclicphotophosphorylation, and the ATP requirements for autotrophicgrowth.     

The Mechanism of Photosynthetic Use of Bicarbonate by Hydrodictyon africanum

Hydrodictyon africanum can photosynthesize at high pH underconditions in which HCO₃^– rather than CO₂ is the carbonspecies entering the cell. A passive entry of HCO₃^– seemsunlikely; a metabolic HCO₃^– pump is proposed. It is possiblethat such a pump is related to a light-dependent reaction specificto the use of HCO₃^–. This reaction is dependent on photosystem2, but appears to be independent of ATP. These characteristicsare similar to those of active lightdependent Cl influx in H.africanum, and suggest a similar energy source for the two pumps.The HCO₃^– pump may be electrogenic.     

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.     

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.

---

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     

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     

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.     

Anionen-Influx,ATP-Spiegel und CO2-Fixierung in Limnophila gratioloides und Chara foetida

Summary Influx of anions (5x10^–4 M Cl^– or SO₄ ^2–) across the plasmalemma, ATP levels and CO₂ fixation in Limnophila and Chara have been measured in a comparative study.In Limnophila, influx, ATP level and CO₂ fixation were progressively reduced by increasing concentrations of carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the light (4000 lux) as well as in the dark. In Chara, not only influx but also ATP levels were much less reduced in the light than in the dark.At 5x10^–4 M external salt concentration the action of light or dark is to change active influx of anions. Thus this study provides strong evidence to support the view that active anion uptake is directly dependent on ATP rather than on electron flow or NADPH. The possible significance of differences in the photophosphorylation systems of various plants is stressed.     

Measurement of Simultaneous Oxygen Evolution and Uptake in Hydrodictyon africanum

Oxygen uptake and evolution in illuminated and darkened cellsof Hydrodictyon africanum have been measured using ¹⁸O₂ massspeetrometry. Under conditions of light and CO₂ saturation forphotosynthesis, light stimulates oxygen uptake more than two-fold.This stimulation is prevented by DCMU but is not affected bycyanide or the uncoupler CCCP. The data are consistent withthe occurrence of a pseudocyclic electron flow and photophosphorylationin vivo in H. africanum; this agrees with data on light-dependentactive phosphate influx in this alga. Part of the light-stimulatedoxygen uptake might be involved in glycolate synthesis by thepathway proposed by Coombs and Whittingham.     

Fatty Acid Synthesis in Hydrilla Chloroplasts

The rates of carboxylation, photophosphorylation and acetate incorporation have been compared in the intact and broken chloroplasts of Hydrilla verticillata Royle leaves in the presence and absence of certain inhibitors and metabolites. The intact chloroplasts showed low rates of photophosphorylation, high rates of carboxylation, and exhibited normal capacity for fatty acid biosynthesis. In broken chloroplasts a drastic decrease was observed in the rates of carboxylation and acetate incorporation. However, the rate of photophosphorylation was considerably increased. In the presence of light, inhibitors such as iodoacetamide, arsenite and sodium azide decreased the photophosphorylation rate. F-1,6-di-P and PGA stimulated CO₂ fixation rate. In the absence of artificial light, inhibitors such as sodium arsenite, gluconate-6-phosphate, sodium azide and iodoacetamide decreased the rate of CO₂ fixation. CoA, ATP, G-6-P, F-1,6-di-P Stimulated the synthesis of fatty acids. Exogenous supply of ADP. NADH, NADP and NADPH did not stimulate fatty acid biosynthesis probably because these compounds could not gain entry into the chloroplasts. Light was necessary for the in vitro fatty acid biosynthesis.     

Fatty acid synthesis by spinach chloroplasts III. Relationship between fatty acid synthesis and photophosphorylation

The modes of actions of photosynthetic inhibitors on photosynthesisand fatty acid synthesis were examined. DCMU, an electron transport inhibitor, inhibited fatty acidsynthesis and photophosphorylation to the same extent, suggestingdependence of fatty acid synthesis on photosynthesis. The samewas also the case with FCCP, a photophosphorylation uncoupler.In contrast, NH₄Cl and phlorizin at concentrations completelysuppressing ATP formation, only partially inhibited the fattyacid synthesis. These facts suggest that a certain level ofhigh-energy intermediate (state) is responsible for the lightenhancement of fatty acid synthesis. This idea is further supportedby the fact that the partial inhibition of fatty acid synthesisby NH₄Cl was relieved by addition of DCCD at low concentrationssuppressing the ATP formation but not completely destroyingthe high energy intermediate. The lag period in the initial period of fatty acid synthesiswas shortened by preillumination of chloroplasts, even in theabsence of ADP. This indicates that the light dependent fattyacid synthesis is closely associated with the high-energy intermediate(state), but not directly with ATP formation by photophosphorylation. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

The stoichiometry (ATP/2e− ratio) of non-cyclic photophosphorylation in isolated spinach chloroplasts

1. The stoichiometry of non-cyclic photophosphorylation and electron transport in isolated chloroplasts has been re-investigated. Variations in the isolation and assay techniques were studied in detail in order to obtain optimum conditions necessary for reproducibly higher ADP/O (equivalent to ATP/2e^?) and photosynthetic control ratios.2. Studies which we carried out on the possible contribution of cyclic phosphorylation to non-cyclic phosphorylation suggested that not more than 10% of the total phosphorylation found could be due to cyclic phosphorylation.3. Photosynthetic control, and the uncoupling of electron transport in the presence of NH₄Cl, were demonstrated using oxidised diaminodurene as the electron acceptor. A halving of the ADP/O ratio was found, suggesting that electrons were being accepted between two sites of energy conservation, one of which is associated with Photosystem I and the other associated with Photosystem II.4. ATP was shown to inhibit State 2 and State 3 of electron transport, but not State 4 electron transport or the overall ADP/O ratio, thus confirming its activity as an energy transfer inhibitor. It is suggested that part of the non-phosphorylating electron transport rate (State 2) which is not inhibited by ATP is incapable of being coupled to subsequent phosphorylation triggered by the addition of ADP (State 3). If the ATP-insensitive State 2 electron transport is deducted from the State 3 electron transport when calculating the ADP/O ratio, a value of 2.0 is obtained.5. The experiments reported demonstrate that there are two sites of energy conservation in the non-cyclic electron transfer pathway: one associated with Photosystem II and the other with Photosystem I. Thus, non-cyclic photophosphorylation can probably produce sufficient ATP and NADPH “in vivo” to allow CO₂ fixation to proceed.     

Volume-sensitive K influx in human red cell ghosts

K influx into resealed human red cell ghosts increases when the ghosts are swollen. The influx demonstrates properties similar to volume-sensitive K fluxes present in other cells. The influx is, for the most part, insensitive to the nature of the major intracellular cation and therefore is not a K-K exchange. The influx is much greater when the major anion is Cl than when the major anion is NO3; Cl stimulates the flux and, at constant Cl, NO3 inhibits it. Increase in the influx rate is rapid when shrunken ghosts are swollen or when NO3 is replaced by Cl. The volume-sensitive K influx requires intracellular MgATP at low concentrations, and ATP cannot be replaced by nonhydrolyzable ATP analogues. The volume-sensitive influx is inhibited by Mg2+ and by high concentrations of vanadate, but is stimulated by low concentrations of vanadate. It is not modified by cAMP, the removal of Ca2+ by EGTA, substances that activate protein kinase C, or by inhibition of phosphatidylinositol kinase. The influx is inhibited by neomycin and by trifluoperazine.