Effects of monovalent cations on light energy distribution between two pigment systems of photosynthesis in isolated spinach chloroplasts

The effects of monovalent cations on the light energy distribution between two pigment systems of photosynthesis were studied in isolated spinach chloroplasts by measuring chlorophyll a fluorescence and photochemical reactions.

The addition of NaCl to the chloroplast suspension produced a 40–80% increase in fluorescence yield measured at 684 nm at room temperature. The fluorescence increase was completed about 5 min after the addition. The effect saturated at 100 mM NaCl. Low-temperature fluorescence spectra showed that NaCl increased the yields of two fluorescence bands of pigment system II at 684 and 695 nm but decreased that of pigment system I at 735 nm. Similar effects on chlorophyll a fluorescence at room and at low temperatures were obtained with NaBr, NaNO₃, Na₂SO₄, LiCl, KCl, RbCl, CsCl, NH₄Cl and CH₃NH₃Cl.

NaCl suppressed the quantum efficiency of NADP⁺ reduction supported by the ascorbate-2,6-dichlorophenolindophenol (DCIP) couple as an electron donor system in the presence of 3-(3′,4′-chlorophenyl)-1,1-dimethylurea (DCMU). On the other hand, NaCl only slightly enhanced the quantum yield of photoreaction II measured by the Hill reaction with DCIP.

It is concluded that the monovalent cations tested suppressed the excitation transfer from pigment system II to pigment system I; the effects were the same as those of alkaline earth metals and Mn²⁺ (refs. 1, 2).     

An NAD(P) reductase derived from Chlorobium thiosulfa-tophilum: Purification and some properties

A highly purified preparation of an NAD(P) reductase was obtained from Chlorobium thiosulfatophilum and some of its properties were studied. The enzyme possesses FAD as the prosthetic group, and reduces benzyl viologen, 2,6-dichloro-phenolindophenol and cytochromes c, including cytochrome c-555 (C. thiosulfato-philum), with NADPH or NADH as the electron donor. It reduces NADP⁺ or NAD⁺ photosynthetically with spinach chloroplasts in the presence of added spinach ferredoxin. It reduces the pyridine nucleotides with reduced benzyl viologen. The enzyme also shows a pyridine nucleotide transhydrogenase activity. In these reactions, the type of pyridine nucleotide (NADP or NAD) which functions more efficiently with the enzyme varies with the concentration of the nucleotide used; at concentrations lower than approx. 1.0 mM, NADPH (or NADP⁺) is better electron donor (or acceptor), while NADH (or NAD⁺) is a better electron donor (or acceptor) at concentrations higher than approx. 1.0 mM. Reduction of dyes or cytochromes c catalysed by the enzyme is strongly inhibited by NADP⁺, 2′-AMP and and atebrin.     

Chlorophyll a fluorescence and photochemical activities of chloroplast fragments

1. Comparative studies were made on the fluorescence characteristics of chlorophyll a at 20° and −193°, and quantum efficiencies for P 700 oxidation and NADP⁺ reduction were measured in chloroplasts and chloroplast fragments obtained after incubation with 0.5% digitonin.

2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a₂). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a₂ from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a₁). The ratio of the fluorescence yields of chlorophyll a₁ and chlorophyll a₂ is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.

3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP⁺ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP⁺ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.

4. The constancy of the fluorescence yield of chlorophyll a₁ in the small fragments, under conditions at which P 700 is oxidized and NADP⁺ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a₁ is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a₁ to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.

5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments.     

Isolation of Photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition

Partition in an aqueous Dextran-polyethylene glycol two-phase system has been used for the separation of chloroplast membrane vesicles obtained by press treatment of a grana-enriched fraction after unstacking in a low salt buffer.

The fractions obtained were analysed with respect to chlorophyll, photochemical activities and ultrastructural characteristics. The results reveal that the material partitioning to the Dextran-rich bottom phase consisted of large membrane vesicles possessing mainly Photosystem II properties with very low contribution from Photosystem I. Measurements of the H₂O to phenyl-p-benzoquinone and ascorbate-Cl₂Ind to NADP⁺ electron transport rates indicate a ratio of around six between Photosystem II and I.

The total fractionation procedure could be completed within 2–3 h with high recovery of both the Photosystem II water-splitting activity and the Photosystem I reduction of NADP⁺.

These data demonstrate that press treatment of low-salt destabilized grana membranes yields a population of highly Photosystem-II enriched membrane vesicles which can be discriminated by the phase system. We suggest that such membrane vesicles originate from large regions in the native grana membrane which contain virtually only Photosystem II.     

Activation of CO2 fixation in isolated spinach chloroplasts

1. 1. The effect of the Mg²⁺ concentration on the CO₂ fixation activity in situ in isolated and intact spinach chloroplasts upon suspension in hypotonic medium was examined. CO₂ fixation in the dark was activated 25–100 fold by 20 mM Mg²⁺ in the presence of added ATP plus either ribulose 5-phosphate or ribose 5-phosphate. 20 mM Mg²⁺-stimulated fixation only 2–3 fold in the presence of the substrate of fixation, ribulose 1,5-diphosphate. The highest Mg²⁺-stimulated rate of fixation in the dark observed with chloroplasts was 480 μmoles CO₂ fixed per mg chlorophyll per h.

2. 2. The concentration of bicarbonate at half of the maximal velocity (apparent K_m) during the Mg²⁺-stimulated fixation of CO₂ was 0.4 mM in the presence of ATP plus ribose 5-phosphate and 0.6 mM with ribulose 1,5-diphosphate.

3. 3. Dithioerythritol or light enhanced Mg²⁺-stimulated CO₂ fixation 1–3 fold in the presence of ATP plus ribose 5-phosphate but not ribulose 1,5-diphosphate.

4. 4. These results indicate that Mg²⁺ fluxes in the stroma of the chloroplast could control the activity of the phosphoribulokinase with a lesser effect on the ribulosediphosphate carboxylase. An increase in Mg²⁺ of 6–10 mM in the stroma region of the chloroplast would be enough to activate CO₂ fixation during photosynthesis.

Improvement in separation of system I and system II particles of photosynthesis obtained by digitonin treatment

By a combined use of digitonin treatment and subsequent centrifugation on a linear sucrose density gradient, the whole green material of the chloroplast lamellae was separated into System I and System II particle fractions, leaving no other fractions of intermediate properties at the final step of separation.

Each of these particle fractions obtained had properties characteristic of System I or System II with respect to the molar ratio of chlorophyll a/chlorophyll b, the content of P700, the fluorescence emission spectrum at −196°;, photoreduction activities with ferricyanide and NADP⁺, and induction of fluorescence.

About 40 and 50% of the total chlorophyll in the original chloroplasts were recovered in System I and System II particles, respectively. Only small amounts of total chlorophyll (less than 10%) were found as free chlorophyll detached from the lamellae through the digitonin treatment.

These results support the view that the lamellae of chloroplasts are composed of about equal amounts of System I and System II particles on a chlorophyll basis.     

Expression of Azotobacter vinelandii soluble transhydrogenase perturbs xylose reductase-mediated conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae

Pyridine nucleotide transhydrogenase is a metabolic enzyme transferring the reducing equivalent between two nucleotide acceptors such as NAD⁺ and NADP⁺ for balancing the intracellular redox potential. Soluble transhydrogenase (STH) of Azotobacter vinelandii was expressed in a recombinant Saccharomyces cerevisiae strain harboring the Pichia stipitis xylose reductase (XR) gene to study effects of redox potential change on cell growth and sugar metabolism including xylitol and ethanol formation. Remarkable changes were not observed by expression of the STH gene in batch cultures. However, expression of STH accelerated the formation of ethanol in glucose-limited fed-batch cultures, but reduced xylitol productivity to 71% compared with its counterpart strain expressing xylose reductase gene alone. The experimental results suggested that A. vinelandii STH directed the reaction toward the formation of NADH and NADP⁺ from NAD⁺ and NADPH, which concomitantly reduced the availability of NADPH for xylose conversion to xylitol catalyzed by NADPH-preferable xylose reductase in the recombinant S. cerevisiae.     

Fluorescence induction and activity of ferredoxin-NADP reductase in Bryopsis chloroplasts

Effects of medium osmolarity on the rate of CO₂ fixation, the rate of the NADP⁺-Hill reaction, and the DPS₁ transient of chlorophyll fluorescence were measured in intact Bryopsis chloroplasts. Upon decreasing the sorbitol concentration from 1.0 M (the isoosmotic conditions) to 0.25 M, the envelopes of the chloroplasts became leaky to small molecules, resulting in a considerable depression of the CO₂-fixation rate and a higher rate of the NADP⁺-Hill reaction whereas the DPS₁ transient was unaffected. This DPS₁ transient of chlorophyll fluorescence is thought to be caused by the photoactivation of electron flow on the reducing side of Photosystem I at a site occurring after ferredoxin and probably before the reduction of NADP⁺ (Satoh, K. and Katoh, S. (1980) Plant and Cell Physiol. 21, 907–916). Little effect of NADP⁺ on the DPS₁ transient and a marked lag in NADP⁺ photo-reduction in dark-adapted (inactivated) chloroplasts support the hypothesis that the site of dark inactivation is prior to the reduction site of NADP⁺, and therefore, that ferredoxin-NADP⁺ reductase is inactivated in the dark and activated in the light. Moreover, at 0.25 M sorbitol, the activity of ferredoxin-NADP⁺ reductase itself (2,6-dichlorophenolindophenol reduction by NADPH) was shown to increase according to dark-light transition of the chloroplasts. At low osmolarities (below 0.1 M sorbitol), the difference in the diaphorase activity between dark-and light-adapted chloroplasts and the lag time observed in the NADP⁺ photoreduction were lowered. This may correspond to a less pronounced DPS₁ transient at low concentrations of sorbitol. The mechanism of the photo-activation is discussed.     

Rapid microfluorimetry of enzyme reactions in single living cells

An optimized photon counting technique allows the microfluorimetric study of NAD⁺ (or NADP⁺) reduction-reoxidation transients in single living cells with a time resolution in the range of 1/50-1/100 sec. The transients resulting from the micro-electrophoretic addition of metabolites (e.g. Glc-6-P or Glc-1-P) can be analyzed in terms of early parameters (e.g. initial lag, rise half time or full rise time) and overall parameters (time of rise and half decay, amplitude, reoxidation time). Both the initial lag and rise half time are considerably longer with Glc-1-P than with Glc-6-P, possibly due to control at the phosphoglucomutase or compartmentation of glycolytic phosphate esters. While glycolytic NAD⁺ (or NADP⁺) reduction proceeds adequately in aerobic EL2 and EAT ascites cells (although ΔNADH/Δt is higher at anaerobiosis), it is critically dependent upon anaerobiosis in L and astrocytoma cells. Thus by rapid microfluorimetry it is possible to resolve the rising phase or other segments of the fluorescence transients into components each corresponding to a particular step in the sequence of intracellular events or control states.     

Wavelength-dependent quantum yield of ATP synthesis and NADP reduction in normal and dichlorodimethylphenylurea-poisoned chloroplast

At short wavelengths (525–690 mμ) the direct measurement of the quantum yield of the photoreduction of NADP⁺ in normal O₂-evolving spinach chloroplasts is constant ( approx. 0.3 equiv/hv). At short wavelengths (<690 mμ) the quantum yield for NADP⁺ reduction in 3(3,4-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts supplied with the ascorbate-2,6-dichlorophenolindophenol couple (donor system) is approx. half as efficient as the normal system. At long wavelengths the quantum yield of NADP⁺ reduction in the donor system increases by a factor of 2 ( approx. 0.3 equiv/hv) when compared with the corresponding yield for the donor system at short wavelengths ( approx. 0.15 equiv/hv).

Between 525 and 690 mμ, the phosphorylation yield for the normal system is constant ( = 0.15 ATP/hv), maintaining a constant P/2e ratio of unity. The P/2e ratios indicate a tight coupling between phosphorylation and electron transport encompassing a single phosphorylation site for the transfer of two electrons.

Between 525 and 680 mμ, the phosphorylation yield for the donor system is constant ( approx. 0.04 ATP/hv), maintaining a P/2e ratio of approx. 0.5. At longer wavelengths (>690 mμ) the phosphorylation yield of the donor system rises ( approx. 0.07–0.08 ATP/hv) concomitant with the rise in the yield of electron flow.

These experiments suggest the possibility that two types of phosphorylation processes operate in chloroplasts, (1) a short-wavelength process coupled to the normal O₂-evolving activity, and (2) a long-wavelength process coupled to the electron-donor activity of reagents such as DCIP.     

Influence de l'oxygene sur les transferts d'electrons de la photosynthese. II. Influence de tres faibles concentrations en oxygene sur la reduction du NADP par les chloroplastes isoles

Influence of oxygen on the electron transfers of photosynthesis. II. Influence of very low oxygen concentration on the NADP⁺ reduction by isolated chloroplasts

The influence of very low O₂ concentration on the NADP⁺ reduction by isolated spinach chloroplasts has been studied.

The results show that in the presence of very low O₂ concentration (< 0.3%) NADP⁺ reduction is partially inhibited. This inhibition may be partially reversed under some conditions, especially when, in spite of the presence of an O₂ trap (glucose plus glucose oxidase (EC 1.1.3.4)) an O₂ evolution is observed.     

Catalysis of plastocyanin electron self-exchange by redox-inert multivalent cations

Electron self-exchange in solutions of the ‘blue’ copper protein plastocyanin is catalysed by the redox-inert multivalent cations Mg²⁺ or Co(NH₃)³⁺₆. Measurements of specific ¹H-NMR line broadening with 50% reduced solutions in the presence of these cations show that electron exchange proceeds through encounters of cation-protein complexes which dissociate at high ionic strength. In the presence of 8mM (5 equivalents/total protein) Co(NH₃)³⁺₆, with 10 mM cacodylate (pH^*6.0) as background electrolyte, the bimolecular rate constant at 25°C is 7 × 10⁴ M⁻¹·s⁻¹. For comparison, the ‘electrostatically screened’ rate constant measured in 0.1 M KCl in the absence of added multivalent cations is ˜ 4 × 10³ M¹·s⁻¹.

Plastocyanin Electron self-exchange NMR Protein-protein interaction Multivalent cation Blue copper protein     

Electron paramagnetic resonance saturation studies of P-700 reaction center chlorophyll in plant photosynthesis

Electron paramagnetic resonance (EPR) power saturation and saturation recovery methods have been used to determine the spin lattice, T₁, and spin-spin, T₂, relaxation times of P-700⁺ reaction-center chlorophyll in Photosystem I of plant chloroplasts for 10 K T 100 K. T₁ was 200 μs at 100 K and increased to 900 μs at 10 K. T₂ was 40 ns at 40 K and increased to 100 ns at 10 K. T₁ for 40 K T 100 K is inversely proportional to temperature, which is evidence of a direct-lattice relaxation process. At T = 20 K, T₁ deviates from the 1/T dependence, indicating a cross relaxation process with an unidentified paramagnetic species. The individual effects of ascorbate and ferricyanide on T₁ of P-700⁺ were examined: T₁ of P-700⁺ was not affected by adding 10 mM ascorbate to digitonin-treated chloroplast fragments (D144 fragments). The P-700⁺ relaxation time in broken chloroplasts treated with 10 mM ferricyanide was 4-times shorter than in the untreated control at 40 K. Ferricyanide appears to be relaxing the P-700⁺ indirectly to the lattice by a cross-relaxation process. The possibility of dipolar-spin broadening of P-700⁺ due to either the iron-sulfur center A or plastocyanin was examined by determining the spin-packet linewidth for P-700⁺ when center A and plastocyanin were in either the reduced or oxidized states. Neither reduced center A nor oxidized plastocyanin was capable of broadening the spin-packet linewidth of the P-700⁺ signal. The absence of diplolar broadening indicates that both center A and plastocyanin are located at a distance at least 3.0 nm from the P-700⁺ reaction center chlorophyll. This evidence supports previous hypotheses that the electron donor and acceptor to P-700 are situated on opposite sides of the chloroplast membrane. It is also shown that the ratio of photo-oxidized P-700 to photoreduced centers A and B at low temperature is 2 : 1 if P-700 is monitored at a nonsaturating microwave power.     

Inhibition of K+ efflux prevents mitochondrial dysfunction, and suppresses caspase-3-, apoptosis-inducing factor-, and endonuclease G-mediated constitutive apoptosis in human neutrophils

Neutrophils die rapidly via apoptosis and their survival is contingent upon rescue from constitutive programmed cell death by signals from the microenvironment. In these experiments, we investigated whether prevention of K⁺ efflux could affect the apoptotic machinery in human neutrophils. Disruption of the natural K⁺ electrochemical gradient suppressed neutrophil apoptosis (assessed by annexin V binding, nuclear DNA content and nucleosomal DNA fragmentation) and prolonged cell survival within 24–48 h of culture. High extracellular K⁺ (10–100 mM) did not activate extracellular signal-regulated kinase (ERK) and Akt, nor affected phosphorylation of p38 MAPK associated with constitutive apoptosis. Consistently, pharmacological blockade of ERK kinase or phosphatidylinositol 3-kinase (PI 3-kinase) did not affect the anti-apoptotic action of KCl. Inhibition of K⁺ efflux effectively reduced, though never completely inhibited, decreases in mitochondrial transmembrane potential (ΔΨ_m) that preceded development of apoptotic morphology. Changes in ΔΨ_m resulted in attenuation of cytochrome c release from mitochondria into the cytosol and decreases in caspase-3 activity. Culture of neutrophils in medium containing 80 mM KCl with the pan-caspase inhibitor Z-VAD-FMK resulted in slightly greater suppression of apoptosis than KCl alone. High extracellular KCl also attenuated translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) from mitochondria to nuclei. The DNase inhibitor, aurintricarboxylic acid (ATA) partially inhibited nucleosomal DNA fragmentation, and the effects of ATA and 80 mM KCl were not additive. These results show that prevention of K⁺ efflux promotes neutrophil survival by suppressing apoptosis through preventing mitochondrial dysfunction and release of the pro-apoptotic proteins cytochrome c, AIF and EndoG independent of ERK, PI 3-kinase and p38 MAPK. Thus, K⁺ released locally from damaged cells may function as a survival signal for neutrophils.     

Coupling factor activity of the purified pea mitochondrial F1-ATPase

The pea cotyledon mitochondrial F₁-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose /2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF₁-ATPase. The molecular weights of pea mitochondrial F₁-ATPase and pea chloroplast CF₁-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F₁-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF₁-ATPase. The purified mitochondrial F₁-ATPase exhibited coupling factor activity. In spite of the observed differences between CF₁ and F₁, the mitochondrial enzyme stimulated ATP formation in CF₁-depleted pea chloroplast membranes. Thus, the mitochondrial F₁ was able to substitute functionally for the chloroplast CF₁ in reconstituting photophosphorylation.     

Pigment systems and electron transport in chloroplasts I. Quantum requirements for the two light reactions in spinach chloroplasts

From studies of electron-transport reactions of isolated spinach chloroplasts, we observe the following quantum requirements: (A) For the photoreduction of NADP⁺, measured both aerobically and anaerobically, in a 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) poisoned system with ascorbate and reduced 2,6-dichlorophenolindophenol (DCIPH₂) present as electron donors, the quantum requirements are 1.0 ± 0.05 at wavelengths longer than 700 nm of actinic light, and 1.5–2.5 for wavelengths between 620 and 680 nm. (B) For the photoreduction of 2,6-dichlorophenolindophenol (DCIP) with water as the electron donor, the quantum requirements are 1.0 ± 0.05 in the range 630–660 nm. (C) For the photoreduction of NADP⁺ with water as the electron donor, the quantum requirements are 2.0 ± 0.1 in the wavelength range 640–678 nm of actinic light, increasing to 6 or greater at wavelengths beyond 700 nm. These results are shown to be inconsistent with the “separate package” model for the two pigment systems in higher plant photosynthetic electron transport. The evidence is most easily interpreted using a “controlled spillover” model, in which the transfer of electronic excitation energy from one pigment system to the other is under the control of incompletely identified factors in the reaction mixture.

At moderate light intensities the steady state rate of the [ascorbate + DCIPH₂ → NADP⁺] reaction (A) in the presence of DCMU and added ferredoxin can be increased more than 3 times when saturating amounts of plastocyanin and ferredoxin-NADP reductase are added to the chloroplasts. Similarly, the steady-state rate of the [H₂O → DCIP] Hill reaction (B) is increased about 3-fold by added MgCl₂ and plastocyanin, but added ferredoxin or ferredoxin-NADP reductase have no effect on this reaction. Plastocyanin appears to be the electron transport component which couples to DCIP, either in the oxidized or in the reduced form, in the reaction media. The steady-state rate of the [H₂O → NADP⁺] reaction (C) with saturating amounts of ferredoxin can be further increased more than 3-fold when MgCl₂, plastocyanin and ferredoxin-NADP reductase are added.     

Chemical modification of the active site of ferredoxin-NADP reductase and conformation of the binary ferredoxin/ferredoxin-NADP reductase complex in solution

Ferredoxin-NADP⁺ reductase (EC 1.18.1.2) was chemically modified by the triplet probe eosin isothiocyanate (eosin-NES). Incorporation of 1 mol eosin-NCS/mol ferredoxin-NADP⁺ reductase completely inhibited binding of NADP⁺/NADPH to the enzyme. Binding of eosin without the reactive group to the enzyme was shown to be reversible but to compete with NADP⁺/NADPH with a K_i of approx. 5 μM. The binding site of eosin-NCS has been located in the primary sequence ferredoxin-NADP⁺ reductase. After specific cleavage of arginine with trypsin a single labelled peptide was obtained and identified as the fragment from residue 179–228 in the primary sequence. Binding of eosin-NCS occurred in either of two predicted helices (residues 179–189 or 212–228) which are both part of an /β structure characteristic for nucleotide binding folds. The rotational diffusion in solution of the eosin-labelled ferredoxin-NADP⁺ reductase and its complex with ferredoxin was measured with laser flash spectroscopy under photoselection. From the measured rotational correlation times and the known structure of ferredoxin-NADP⁺ reductase at 3.7 Å resolution, we propose that ferredoxin is bound to ferredoxin-NADP⁺ reductase between the two domains of the flavoprotein. The two ferredoxin-NADP⁺ reductase domains and ferredoxin form a triangle which results in a highly integrated binary complex.     

Diflubenzuron affects gamma-thioGTP stimulated Ca transport in vitro in intracellular vesicles from the integument of the newly molted american cockroach, Periplanet americana L.

To study the mechanism of action of diflubenzuron (DFB) and other benzoylphenylureas, we have initially hypothesized that their action may be related to exocytosis: to test the hypothesis, we obtained an intracellular vesicle preparation from the homogenate of integument of newly molted American cockroachs (Periplaneta americana L.) in 10 mM MES buffer containing 250 mM sucrose (isotonic) and 2.5 mM MgSO₄, at pH 6.6. By studying DFB's effect on various ion transporting activities, we demonstrated that calcium uptake in this intracellular particulate preparation was significantly inhibited by DFB at low concentrations (e.g., 10⁻⁸ M). Such an inhibitory effect of DFB on Ca²⁺ uptake was eliminated by the addition of ionophores or membrane disruptors, as well as the sonication of vesicle preparation. On the other hand, oligomycin, protein phosphorylation modulators, Na⁺, and Li⁺ did not affect the calcium uptake. Among ionophores, agents disrupting H⁺ gradients (e.g. FCCP and NEM) totally eliminated ⁴⁵Ca uptaking activity by vesicles as well as the inhibitory effect of DFB. Among calcium ion modulators, calmodulin inhibitors such as calmidazolium and trifluoperazine decreased the Ca²⁺-uptake, whereas membrane calcium channel blocker, verapamil, did not. ATP and γ-S-GTP stimulated Ca²⁺ uptake. However, the former increased only the DFB insensitive portion and the latter largely the DFB sensitive part of Ca²⁺. Together these data support the hypothesis that the action site of DFB in this preparation is the GTP-dependent Ca²⁺ transport process which is coupled to vacuolar type intracellular vesicles in the integument cells.     

NADPH/NADP ratios in photosynthesizing reconstituted chloroplasts

Levels of reduced and oxidized triphosphopyridine nucleotides have been determined in reconstituted spinach chloroplasts and compared with levels in whole isolated chloroplasts during photosynthesis and darkness. The ratio of NADPH/NADP⁺ reaches values slightly above 1.0 at the beginning of photosynthesis, less than half the ratio attained with whole chloroplasts. Nonetheless these lower ratios are sufficient to maintain high rates of photosynthetic carbon dioxide fixation and reduction, which are comparable in the reconstituted chloroplasts to the rates found with whole chloroplasts. As with whole chloroplasts there is a decline in the ratio of NADPH/NADP⁺ as a function of time of photosynthesis. The effect of addition of bicarbonate (6 mM) in causing a transient drop in the ratio of NADPH/NADP⁺ is described and discussed in terms of the reversibility of the reduction of 3-phosphoglycerate to triose phosphate. The ratio NADPH/NADP⁺ can be improved by the addition of more lamellae either before or during the course of photosynthesis, and this improvement in ratio is accompanied by an improved rate of CO₂ fixation or a more sustained rate of CO₂ fixation with time of photosynthesis. The importance of NADPH/NADP⁺ ratio not only to the reduction of 3-phosphoglycerate to triose phosphate but also to the activation of the ribulose-1,5-diphosphate carboxylasemediated step is discussed.     

The mechanism of photosynthetic sulfate reduction by isolated chloroplasts

The reduction of sulfate by isolated spinach chloroplasts was studied. A reconstituted system of broken chloroplasts and of chloroplast extract reduced sulfate to sulfite in the light when ADP, NADP⁺, ferredoxin and glutathione were added. The chloroplast extract reduced sulfate to sulfite in the dark if supplemented with ATP and with reduced glutathione. Neither ferredoxin nor NADPH were needed for this reduction in the dark.

A sulfite reductase was purified from spinach leaves. Broken chloroplasts and sulfite reductase reduced sulfite to sulfide in the light when ferredoxin was added. NADP⁺ was not required for this reduction.

The results suggest that in chloroplasts a sulfate activated by ATP (phosphoadenosine phosphosulfate) is reduced to sulfite by a sulfhydryl compound and that sulfite is reduced to sulfide by a ferredoxin-dependent sulfite reductase.