Light-induced rapid absorption changes during photosynthesis. VII. Some reactions in sonicated chloroplasts

1. Spinach chloroplasts subjected to sonication show light-induced absorption changes at 700 mμ characteristic of the photooxidation of the chlorophyll component P700. The appearance of P700 absorption changes probably resulted from the release of plastocyanin thus interrupting the electron flow between pigment systems 1 and 2. The general features of the absorption-change transients are similar to those observed previously with digitonin-treated chloroplasts. The addition of 2 mM ascorbate or 10 μM 3-(3,4-dichlorophenyl)-1, 1-dimethylurea had practically no effect on either the magnitude or the dark decay of the transient absorption change.

2. Phenazine methosulfate (PMS) (in the presence or in the absence of ascorbate) reduction appeared to be coupled to P700 photooxidation, as shown by the corresponding transients at 430 and 388 mμ. The absorbance changes at these two wavelengths indicate that the amount of PMS photoreduced was equivalent to that of P700 photooxidized. Higher PMS concentrations accelerate the dark decay of the P700 signal. When PMS alone is present, anaerobiosis caused the dark decay to become more rapid than in the presence of ascorbate.

3. Unlike PMS, other redox agents such as 2,6-dichlorophenolindophenol, N,N,N′,N′-tetramethyl-p-phenylenediamine or diaminodurol in the presence of excess ascorbate, did not noticeably affect the kinetics of the dark decay at 430 or 703 mμ, suggesting that these reduced species are not efficiently coupled to photooxidized P700.

4. The onset and decay rates of the P700 transient in the presence of PMS and excess ascorbate was insensitive to temperature between 25° and o°. However, when the chloroplast sample was frozen at temperatures ranging from −5° to −196°, all reactions ceased. When the frozen (−196°) sample was brought back to the room temperature, the reaction was restored completely. Fresh broken chloroplasts behave similarly. Digitonin-treated chloroplasts persisted down to about −25° but with diminishing magnitude and slower decay.     

Photoaccumulation of two ascorbyl free radicals per photosystem I at 200 K

Illumination of photosystem I (PSI) from the cyanobacterium Synechocystis sp. PCC 6803 at 200 K in the presence of ascorbate leads to the formation of two ascorbyl radicals per PSI, which are formed by P700(+) reduction by ascorbate. During photoaccumulation, one half of the ascorbyl radicals is formed with a halftime of 1 min and the other half with a halftime of 7 min. Pulsed electron paramagnetic resonance (EPR) experiments with protonated/deuterated PSI show that a PSI proton/deuteron is strongly coupled to the ascorbyl radical. Our data indicate that reactive ascorbate molecules bind to PSI at two specific locations, which might be symmetrically located with respect to the pseudo-C(2) axis of symmetry of the heterodimeric core of PSI. Reduction of P700(+) by ascorbate leads to multiple turnover of PSI photochemistry, resulting in partial photoaccumulation of the doubly reduced species (F(A)(-), F(B)(-)). A modified form of F(B)(-)-in accordance with Chamorovsky and Cammack [Biochim. Biophys. Acta 679 (1982) 146-155], but not of F(A)(-), is observed by EPR after illumination at 200 K, which indicates that reduction of F(B) at 200 K is followed by some relaxation process, in line with this cluster being the most exposed to the solvent.     

Regulation of cyclic electron transport through photosystem I in cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 mutants deficient in respiratory dehydrogenases

The rate of PSI mediated cyclic electron transport was studied in wild type and mutant cells of Synechocystis sp. PCC 6803 deficient in NDH-1 (M55) or succinate dehydrogenase (SDH⁻) that are responsible for the dark reduction of the plastoquinone pool. Kinetics of P700 photooxidation and P700⁺ dark reduction in the presence of 5·10⁻⁵ M 3-(3,4-dichlorophenyl)-1,1-dimethylurea have been registered as light induced absorbance changes at 810 nm resulting from illumination of cells with 730-nm actinic light for 1 sec. It is shown that in the absence of dehydrogenases the rate of dark reduction of P700⁺ in both mutants did not decrease but even increased in NDH-1-less mutant cells as compared with the rate in wild type cells. Dibromothymoquinone drastically reduced the rate of P700⁺ dark reduction both in wild type and in mutant cells. Thus, the cyclic electron transfer from ferredoxin through the plastoquinone pool to P700⁺, which is independent from dehydrogenases, takes place in all the types of cells. Preillumination of cells of wild type and both mutants for 30 min or anaerobic conditions resulted in delay of P700 photooxidation and acceleration of P700⁺ dark reduction, while the level of photosynthesis and respiration terminal acceptors (NAD(P)⁺ and oxygen) decreased. It appears that the rate of P700 photooxidation and P700⁺ dark reduction in cyclic electron transport in Synechocystis wild type and mutant cells is determined by the level of NADP⁺ and oxygen in stroma. A possible approach to evaluation of the levels of these acceptors in vivo is proposed, based on kinetic curve parameters of P700 photoconversions induced by 730-nm light with 1-sec duration.     

Interaction of ascorbate with photosystem I

Ascorbate is one of the key participants of the antioxidant defense in plants. In this work, we have investigated the interaction of ascorbate with the chloroplast electron transport chain and isolated photosystem I (PSI), using the EPR method for monitoring the oxidized centers \( {\text{P}}_{700}^{ + } \) and ascorbate free radicals. Inhibitor analysis of the light-induced redox transients of P₇₀₀ in spinach thylakoids has demonstrated that ascorbate efficiently donates electrons to \( {\text{P}}_{ 7 0 0}^{ + } \) via plastocyanin. Inhibitors (DCMU and stigmatellin), which block electron transport between photosystem II and Pc, did not disturb the ascorbate capacity for electron donation to \( {\text{P}}_{700}^{ + } \) . Otherwise, inactivation of Pc with CN^? ions inhibited electron flow from ascorbate to \( {\text{P}}_{700}^{ + } \) . This proves that the main route of electron flow from ascorbate to \( {\text{P}}_{700}^{ + } \) runs through Pc, bypassing the plastoquinone (PQ) pool and the cytochrome b ₆ f complex. In contrast to Pc-mediated pathway, direct donation of electrons from ascorbate to \( {\text{P}}_{700}^{ + } \) is a rather slow process. Oxidized ascorbate species act as alternative oxidants for PSI, which intercept electrons directly from the terminal electron acceptors of PSI, thereby stimulating photooxidation of P₇₀₀. We investigated the interaction of ascorbate with PSI complexes isolated from the wild type cells and the MenB deletion strain of cyanobacterium Synechocystis sp. PCC 6803. In the MenB mutant, PSI contains PQ in the quinone-binding A₁-site, which can be substituted by high-potential electron carrier 2,3-dichloro-1,4-naphthoquinone (Cl₂NQ). In PSI from the MenB mutant with Cl₂NQ in the A₁-site, the outflow of electrons from PSI is impeded due to the uphill electron transfer from A₁ to the iron-sulfur cluster F_X and further to the terminal clusters F_A/F_B, which manifests itself as a decrease in a steady-state level of \( {\text{P}}_{700}^{ + } \) . The addition of ascorbate promoted photooxidation of P₇₀₀ due to stimulation of electron outflow from PSI to oxidized ascorbate species. Thus, accepting electrons from PSI and donating them to \( {\text{P}}_{700}^{ + } \) , ascorbate can mediate cyclic electron transport around PSI. The physiological significance of ascorbate-mediated electron transport is discussed.     

Effects of oxygen and photosynthesis carbon cycle reactions on kinetics of P700 redox transients in cyanobacterium <Emphasis Type="Italic">Arthrospira platensis</Emphasis> cells

Effects of oxygen and photosynthesis and respiration inhibitors on the electron transport in photosystem I (PSI) of the cyanobacterium Arthrospira platensis cells were studied. Redox transients of P700 were induced by illumination at 730 nm and monitored as kinetics of the absorption changes at 810 nm; to block electron influx from PSII, the measurements were performed in the presence of 30 microM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Inhibitors of terminal oxidases (potassium cyanide and pentachlorophenol) insignificantly influenced the fast oxidation of P700 under aerobic conditions, whereas removal of oxygen significantly decelerated the accumulation of P700(+). In the absence of oxygen the slow oxidation of P700 observed on the first illumination was accelerated on each subsequent illumination, suggesting an activation of the carbon cycle enzymes. Under the same conditions, pentachlorophenol (an uncoupler) markedly accelerated the P700 photooxidation. Under anaerobic conditions, potassium cyanide (an inhibitor of carbon dioxide assimilation) failed to influence the kinetics of redox transients of P700, whereas iodoacetamide (an inhibitor of NADP(H)-glyceraldehyde-3-phosphate dehydrogenase) completely prevented the photooxidation of P700. Thus, the fast photooxidation of P700 in the A. platensis cells under aerobic conditions in the presence of DCMU was caused by electron transport from PSI onto oxygen, and complicated transient changes in the P700 photooxidation kinetics under anaerobic conditions (in the presence of DCMU) were due to involvement of NADP+ generated during the reducing phase of the carbon cycle.     

Effect of detergents on the reliability of a chemical assay for P-700

A chemical assay for P-700 was developed using 0.36 mM potassium ferricyanide as oxidant and 1.6 mM sodium ascorbate as reductant. The major difference from other chemical assays for P-700 is procedural. The method is designed to take advantage of the availability of microprocessor-linked spectrophotometers to obtain greater accuracy by minimizing the spectral changes due to irreversibly oxidized antenna chlorophyll molecules. The value measured for the P-700 concentration in a sample of chloroplasts was not changed by the presence of EDTA, Mg²⁺ or sucrose in the assayed solution. Similarly, half of the detergents tested (Triton X-100, Nonidet P-40, digitonin, Deriphat 160, Miranol S2M-SF and Miranol M2M) did not alter the value when added to the chloroplasts. The remainder of the detergents examined caused a significant decrease or increase in the value for P-700 content. Sodium dodecyl sulfate, of particular interest due to its widespread use, caused a doubling in the amount of apparent P-700. This effect may be due to this detergent and some others enabling an additional long wavelength form of chlorophyll, possibly an intermediary electron acceptor in Photosystem I, to be chemically oxidized and reduced under the assay conditions.     

Properties of the primary electron acceptor complex of photosystem I in the blue green alga Chlorogloea fritschii.

Photooxidation of P700 at low temperatures in membrane fractions from the blue-green alga $\text{Chlorogloea fritschii}$ may be coupled irreversibly to the reduction of a bound ferredoxin. If this ferredoxin is reduced before freezing, P700 photooxidation at low temperatures becomes reversible. This reversible photooxidation is coupled to the reduction of a component with an EPR signal at g = 2.08, 1.88 and 1.78. A complete spectrum of this component has been obtained for the first time. We propose that as in higher plants this component is the primary electron acceptor of Photosystem I, the bound ferredoxin is a secondary electron acceptor. Using ⁵⁷Fe enriched preparations we have shown that the ERP signals attributed to the bound ferredoxin are due to iron containing centres. This experiment did not show the presence of iron in the primary electron acceptor.     

How does iron deficiency disrupt the electron flow in photosystem I of lettuce leaves?

The changes observed photosystem I activity of lettuce plants exposed to iron deficiency were investigated. Photooxidation/reduction kinetics of P700 monitored as ΔA₈₂₀ in the presence and absence of electron transport inhibitors and acceptors demonstrated that deprivation in iron decreased the population of active photo-oxidizable P700. In the complete absence of iron, the addition of plant inhibitors (DCMU and MV) could not recover the full PSI activity owing to the abolition of a part of P700 centers. In leaves with total iron deprivation (0 μM Fe), only 15% of photo-oxidizable P700 remained. In addition, iron deficiency appeared to affect the pool size of NADP⁺ as shown by the decline in the magnitude of the first phase of the photooxidation kinetics of P700 by FR-light. Concomitantly, chlorophyll content gradually declined with the iron concentration added to culture medium. In addition, pronounced changes were found in chlorophyll fluorescence spectra. Also, the global fluorescence intensity was affected. The above changes led to an increased rate of cyclic electron transport around PSI mainly supported by stromal reductants.     

Modulating Effect of Far-Red Light on Activities of Alternative Electron Transport Pathways Related to Photosystem I

Barley (Hordeum vulgare L.) leaves were irradiated with far-red (FR) light of various intensities after different periods of dark adaptation in order to investigate activities of alternative electron transport pathways related to photosystem I (PSI). Photooxidation of P700, the primary electron donor of PSI, was saturated at FR light intensity of 0.15 μmol quanta/(m² s). As the photon flux density was raised in this range, the slow and middle components in the kinetics of P700⁺ dark reduction increased, whereas the fast component remained indiscernible. The amplitudes of the slow and middle components diminished upon further increase of FR photon flux density in the range 0.15–0.35 μmol quanta/(m² s) and remained constant at higher intensities. The fast component of P700⁺ reduction was only detected after FR irradiation with intensities above 0.15 μmol quanta/(m² s); the light-response curve for this component was clearly sigmoid. In dark-adapted barley leaves, three stages were distinguished in the kinetics of P700 photooxidation, with the steady state for P700⁺ achieved within about 3 min. In leaves predarkened for a short time, the onset of FR irradiation produced a very rapid photooxidation of P700. As the duration of dark exposure was prolonged, the amplitude of the first peak in the kinetic curve of photoinduced P700 photooxidation was diminished and the time for attaining the steady-state oxidation level was shortened. After a brief dark adaptation of leaves, ferredoxin-dependent electron flow did not appreciably contributed to the kinetics of P700⁺ dark reduction, whereas the components related to electron donation from stromal reductants were strongly retarded. It is concluded that FR light irradiation, selectively exciting PSI, suffices to modulate activities of alternative electron transport routes; this modulation reflects the depletion of stromal reductants due to continuous efflux of electrons from PSI to oxygen under the action of FR light. __________ Translated from Fiziologiya Rastenii, Vol. 52, No. 6, 2005, pp. 805–813. Original Russian Text Copyright ? 2005 by Egorova, Drozdova, Bukhov.     

Chemical platinization and its effect on excitation transfer dynamics and P700 photooxidation kinetics in isolated photosystem I.

Isolated photosystem I (PSI) reaction center/core antenna complexes (PSI-40) were platinized by reduction of [PtCl6]2- at 20 degrees C and neutral pH. PSI particles were visualized directly on a gold surface by scanning tunneling microscopy (STM) before and after platinization. STM results showed that PSI particles were monomeric and roughly ellipsoidal with major and minor axes of 6 and 5 nm, respectively. Platinization deposited approximately 1000 platinum atoms on each PSI particle and made the average size significantly larger (9 x 7 nm). In addition to direct STM visualization, the presence of metallic platinum on the PSI complexes was detected by its effect of actinic shading and electrostatic shielding on P700 photooxidation and P700+ reduction. The reaction centers (P700) in both platinized and nonplatinized PSI-40 were photooxidized by light and reduced by ascorbate repeatedly, although at somewhat slower rates in platinized PSI because of the presence of platinum. The effect of platinization on excitation transfer and trapping dynamics was examined by measuring picosecond fluorescence decay kinetics in PSI-40. The fluorescence decay kinetics in both platinized and control samples can be described as a sum of three exponential components. The dominant (amplitude 0.98) and photochemically limited excitation lifetime remained the same (16 ps) before and after platinization. The excitation transfer and trapping in platinized PSI-40 was essentially as efficient as that in the control (without platinization) PSI. The platinization also did not affect the intermediate-lifetime (400-600 ps) and long-lifetime (> 2500 ps) components, which likely are related to intrinsic electron transport and to functionally uncoupled chlorophylls, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat-stabilities of Electron Transport Related to Photosystem I in a Thermophilic Blue-green Alga, Synechococcus sp.

Heat-stabilities of photosystem I reactions in a thermophilicblue-green alga, Synechococcus sp. were studied. All the reactionsexamined were highly resistant to heat as compared with thosein ordinary higher plants and algae. Cyt c-553 photooxidation in vivo was abolished by treatmentat 75?C for 5 min. By contrast, P700 photooxidation was extremelyresistant to heat and could not be completely inactivated bytreatment of the cells or isolated thylakoids at about 100?Cfor 5 min. Photooxidation of added Cyt c-553 by isolated thylakoidmembranes was more heat-stable than was this activity in cells.This suggests that heat-treatment caused a perturbation in thestructural integrity of the membranes which is required forefficient electron transfer from Cyt c-553 to P700 in situ. At higher temperatures, the inactivation of Cyt c-553 photooxidationin the membranes parallels the decrease in the rate of P700photooxidation. Spectrophotometric studies with short flashesindicated that inactivation of the electron transport from Cytc-553 to methyl viologen is due to the denaturation of a secondaryelectron acceptor of photosystem I, A₂, and possibly anotheracceptor, P430. ¹ Present address: The Solar Energy Research Group, the Instituteof Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi,Saitama 351, Japan. (Received September 28, 1981; Accepted December 21, 1981)     

Redox states of photosystems I and II in irradiated leaves of wheat seedlings grown under different conditions of nitrogen nutrition

Changes in the redox states of photosystem I (PSI) and PSII in irradiated wheat leaves were studied after growing seedlings on a nitrogen-free medium or media containing either nitrate or ammonium. The content of P700, the primary electron donor of PSI was quantified using the maximum magnitude of absorbance changes at 830 nm induced by saturating white light. The highest content of P700 in leaves was found for seedlings grown on the ammonium-containing medium, whereas its lowest content was observed on seedlings grown in the presence of nitrate. At all irradiances of actinic light, the smallest accumulation of reduced Q_A was observed in leaves of ammonium-grown plants. Despite variations in light-response curves of P700 photooxidation and Q_A photoreduction, the leaves of all plants exposed to different treatments demonstrated similar relationships between steady-state levels of P700⁺ and Q_A ^–. The accumulation of oxidized P700 up to 40% of total P700 content was not accompanied by significant Q_A photoreduction. At higher extents of P700 photooxidation, a linear relationship was found between the steady-state levels of P700⁺ and Q_A ^–. The leaves of all treatments demonstrated biphasic patterns of the kinetics of P700⁺ dark reduction after irradiation by far-red light exciting specifically PSI. The halftimes of corresponding kinetic components were found to be 2.6–4 s (fast component) and 17–22 s (slow component). The two components of P700⁺ dark reduction were related to the existence of two PSI populations with different rates of electron input from stromal reductants. The magnitudes of these components differed for plants grown in the presence of nitrate, on the one hand, and plants grown either in the presence of ammonium or in the absence of nitrogen, on the other hand. This indicates the possible influence of nitrogen nutrition on synthesis of different populations of PSI in wheat leaves. The decrease in far-red light irradiance reduced the relative contribution of the fast component to P700⁺ reduction. The fast component completely disappeared at low irradiances. This finding indicates that the saturating far-red light must be applied to determine correctly the relative content of each PSI population in wheat leaves.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 165–171.Original Russian Text Copyright © 2005 by Dzhibladze, Polesskaya, Alekhina, Egorova, Bukhov.This revised version was published online in April 2005 with a corrected cover date.     

Light-induced changes of b-type cytochromes in the electron transport chain of Euglena chloroplasts

Light-induced changes of b-type cytochromes in Euglena chloroplastswere studied spectrophotometrically.

1. In the dark and at pH 6.5, most of the cytochrome 558 in chloroplastswas in the reduced state, and most of the cytochrome 563, inthe oxidized state. Illumination of chloroplasts at pH 6.5 induceda rapid, but slight oxidation of cytochrome 552 and cytochrome558. The magnitude of photooxidation of cytochrome 558 was greatlyenhanced by the addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea(DCMU). The rate of photooxidation in the presence of DCMU wasstimulated by the addition of 0.15 µM Euglena cytochrome552, or 100 µM methyl viologen.
2. Euglena chloroplasts,incubated at 55°C for 5 min showedno significant absorbancechanges for about 10 min after theonset of illumination. However,greater photooxidation of cytochrome558 was observed afterprolonged illumination, or in the presenceof DCMU or ethylenediaminetetraaceticacid (EDTA). Similar resultswere obtained with chloroplastspre-treated at pH 9.0–10.0for 5 min.
3. At pH 9.5, andin the dark, both cytochrome 563 and cytochrome558 were inan almost reduced state. On illumination at thispH, both cytochromeswere photooxidized, with a complicatedkinetics, showing aninitial rapid and small absorbance decrease,followed by a stagnantphase of temporary retarded reaction.In the presence of DCMUor EDTA, photooxidation proceeded rapidlywithout a stagnantphase.
4. At pH 6.5 cytochrome 558, on cessation of illumination,wasquickly reduced to the initial level. At pH 9.5, there wasalsoappreciable re-reduction of cytochrome 558 and 563 whenthelight was turned off at an early stage of illumination.Theamounts of re-reduction of the cytochromes in the subsequentdark period, however, decreased as photooxidation of cytochromesproceeded. This decrease was accelerated by the presence ofDCMU.
5. At pH 9.5 ascorbate and manganese served as electrondonorsfor die DCMU-sensitive photooxidation of cytochromes558 and563.
6. Experimental results are discussed with specialreference tothe occurrence of two pools of electron carriers,one at thereducing side and the other at the oxidizing sideof photosystem2. The role of manganese in the latter pool ofelectron carriersis also discussed.

(Received March 11, 1970; )     

Elevation of the cyclic GMP concentration in human platelets by sodium ascorbate and 5-hydroxytryptamine.

Sodium L-ascorbate (ascorbate) and sodium D-ascorbate produced a dose-related rise of guanosine 3':5'-cyclic monophosphate (cGMP) in platelets with a maximum increment averaging 25-fold at 5 mM ascorbate. The ascorbate-induced increment in cGMP reached a peak after 1 min and was maintained for 1 h in the presence of ascorbate. 5-hydroxytryptamine (5-HT) also produced a dose-related rise of cGMP in platelets with a peak effect of approximately 25-fold at 16 micrometer 5-HT. The elevation of cGMP in platelets by both ascorbate and 5-HT did not require extracellular calcium and was blocked by inhibitors of cyclo-oxygenase such as aspirin or indomethacin. A maximum ascorbate-induced rise in platelet cGMP at the time of addition of epinephrine, collage or thrombin did not augment the release of [14C]5-hydroxytryptamine ([14C]5-HT) measured over 30 min. Although ascorbate appeared to increase platelet cGMP by modulation of endoperoxide formation, its failure to aggregate platelets or to influence the release reaction indicates that the ascorbate-stimulated rise in cGMP does not have a simple relationship to thromboxane formation.     

Correlation of reaction-center chlorophyll (P-700) oxidation and bound iron-sulfur protein photoreduction in chloroplast Photosystem I at low temperatures

The extent of P-700 photooxidation at 18 °K has been followed in three different chloroplast preparations (unfractionated chloroplasts and two preparations enriched in Photosystem I). More than 90% of P-700⁺ formation in all preparations was eliminated by the addition of sodium dithionite at pH 10. Photoreduction of a bound chloroplast iron-sulfur protein was also decreased by at least 90% under similar conditions. Electron paramagnetic resonance spectra of the chloroplast preparations in the presence of dithionite showed chemical reduction of bound iron-sulfur protein under conditions where primary photochemistry is eliminated. These results indicate that P-700 photooxidation is concomitant with photoreduction of a bound iron-sulfur protein and that this iron-sulfur protein functions as the primary electron acceptor of Photosystem I.     

Dependence of chlorophyll P700 redox transients during the induction period on the transmembrane distribution of protons in chloroplasts of pea leaves

Differential absorbance measurements and fluorometry were applied to examine the impact of dicyclohexylcarbodiimide (DCCD, an inhibitor of H⁺ conductance in thylakoid membranes) and nigericin (a K⁺/H⁺ antiporter) on photoinduced redox state transients of chlorophyll P700 and the induction curves of chlorophyll fluorescence in pea (Pisum sativum L., cv. Premium) leaves. The treatment of leaves with DCCD strongly modified the kinetics of P700⁺ absorbance changes (ΔA ₈₁₀) by promoting rapid photooxidation of P700. These characteristic changes in ΔA ₈₁₀ induction kinetics and P700⁺ accumulation did not appear when the leaves were treated with DCCD in the presence of nigericin. In addition to opposite modifications of ΔA ₈₁₀ kinetics evoked by permeability-modifying agents, the fluorescence induction curves differed conspicuously depending on leaf incubation in DCCD solutions with or without nigericin. The observed modifications of fluorescence induction curves and ΔA ₈₁₀ indicate that DCCD suppresses electron transport from photosystem II (PSII) to P700, whereas this inhibition is removed by nigericin. The results suggest that slowing down of the electron transport rate in the presence of DCCD was caused by elevation of ΔpH in thylakoids. The prevention of pH gradient formation in the presence of protonophore lowered also the steady-state P700⁺ level in far-red irradiated leaves and accelerated the subsequent dark reduction of P700. These findings indicate that PSI-driven cyclic electron flow is accelerated after the removal of the pH gradient.     

Dual roles of ubiquinone as primary and secondary electron acceptors in light-induced electron transfer in chromatophores of Chromatium D.

The effects of isooctane-extraction on the quantum yield ofphotooxidation of cytochromes in chromatophores of Chromatiumvinosum, strain D, were investigated. The initial rate of photooxidation of cytochrome c-555 in theisooctane-extracted chromatophores was decreased by repeatedor prolonged preillumination in the presence of 30 mM ascorbate.The minimum number of light quanta absorbed during preilluminationto cause the maximum decrease in the photooxidation of cytochromec-555 was about 2% of the number of bacteriochlorophyll moleculespresent. In the absence of ascorbate no lowering of the initial rateof cytochrome photooxidation was observed after prolonged orrepeated illumination. No decrease in the initial rate due topreillumination was observed in lyophilized or ubiquinone-readdedchromatophores. The initial rate of photooxidation of both the cytochromes c-555and c-552 in partially isooctane-extracted chromatophores (50–90%extraction of ubiquinone) was also decreased by repeated orprolonged illumination in the presence of 30 mM ascorbate. Our previous and present studies indicate that about 10% ofthe total ubiquinone- 7 functions as the primary electron acceptorfor the photooxidation of cytochrome c-552, and that the majorpart of the ubiquinone functions as the common secondary electronacceptor for the photooxidation of cytochromes c-555 and c-552in Chromalium chromatophores. Therefore, ubiquinone probablyhas dual roles in the light-induced electron transfer of Chromatiumchromatophores. (Received July 23, 1975; )     

Na+-linked active transport of ascorbate into cultured bovine retinal pigment epithelial cells: heterologous inhibition by glucose

The transport of ascorbate into cultured bovine retinal pigment epithelial (RPE) cells is reported. Primary or subcultured RPE cells were incubated in the presence of 10-500 microM L-[carboxyl-14C]-ascorbate for various periods of time. Accumulation of ascorbate into RPE cells followed a saturable active transport with a Km of 125 microM and a Vmax of 28 pmole/micrograms DNA/min. RPE intracellular water was calculated to be 0.8 pL/cell, and the transported cellular ascorbate concentration was 7.5 +/- 0.8 mM. Replacement of 150 mM NaCl in the incubation media with choline-Cl strongly inhibited (80 +/- 8%) ascorbate uptake into cultured RPE cells. Although the depletion of cellular ATP by 2,4-dinitrophenol and the inhibition of Na+-K+-ATPase by ouabain reduced ascorbate transport into RPE significantly, active transport of ascorbate was not entirely inhibited by these metabolic inhibitors. The ascorbate analogue, D-isoascorbate, competitively inhibited ascorbate transport into cultured RPE with a Ki of 12.5 mM. Cells grown in the presence of 5 to 50 mM alpha-D-glucose in the growth media did not differ in their ability to transport ascorbate. In contrast, the presence of alpha-D-glucose or its nonmetabolizable analogues, 3-0-methyl-glucose, alpha-methyl-glucose, and 2-deoxy-glucose, but not L-glucose or beta-D-fructose, in the incubation media inhibited ascorbate transport. myo-Inositol (10 or 20 mM) also inhibited ascorbate transport into RPE cells. The active uptake of ascorbate into cultured RPE cells was primarily coupled to the movement of sodium ion down its electrochemical gradient. A bifunctional, cotransport carrier possessing an ascorbate-binding site and a sodium-binding site may be involved in the ascorbate uptake system. The inhibition of ascorbate uptake by sugars appeared to be heterologous in nature, occurring between two distinct carrier systems, both of which were dependent on the sodium ions.