The reactivation of nitrate reductase from spinach (Spinacea oleracea L.) inactivated by NADH and cyanide: effects of peroxidase and associated systems

Nitrate reductase of spinach (Spinacea oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH₂-dependent activity. Ferricyanide, a purely chemical oxidant, produced rapid maximal reactivation (100%) which was 90% complete in less than 3 min. Reactivation occurred slowly and less completely (30–75% in 30 or 60 min) when the enzyme was incubated with pure horseradish peroxidase alone, depending on using one or 20 units and time. Addition of glucose and glucose oxidase to generate hydrogen peroxide increased reactivation slightly (10–15%) with 20 units of peroxidase but more (30–50%) with one unit and to 75–90% of ferricyanide values. Adding catalase decreased reactivation by more than half either with or without glucose oxidase. Glucose and glucose oxidase alone did not cause reactivation. Addition of superoxide dismutase increased reactivation from 50–75% of ferricyanide values with one unit of peroxidase alone but had no effect on greater reactivation obtained in the presence of glucose oxidase. The addition of p-cresol and manganese together increased reactivation with one unit of peroxidase and in the presence of glucose oxidase by about double, but omission of manganese had no effect. However, as shown previously, although trivalent manganese was formed, the residual presence of manganous ions inhibited reactivation. Nevertheless, peroxidase systems either alone or with additionally generated hydrogen peroxide can induce substantial reactivation of nitrate reductase in physiologically relevant conditions.Abbreviations EDTA ethylenediaminetetraacetic acid - FMN flavine mononucleotide     

Some effects of inorganic phosphate on O2 evolution by isolated chloroplasts

1. After an initial lag, isolated spinach chloroplasts evolved O₂ in illuminated reaction mixtures containing bicarbonate but no added phosphate. This evolution soon ceased but could be restarted by the addition of phosphate.

2. The phosphate requirement could be met by orthophosphate, inorganic pyrophosphate, ATP or ADP but not by AMP. Approx. 3 molecules of O₂ were evolved for each molecule of orthophosphate added and approx. 6 for each molecule of pyrophosphate.

3. With CO₂ as the sole added substrate the extent of the initial lag in O₂ evolution was not greatly affected by small quantities of added orthophosphate but as the concentration of orthophosphate was increased there was a progressive increase in the lag and a progressive decrease in the maximum rate. Pyrophosphate failed to produce these effects at a 100 times the concentration and in the presence of pyrophosphate the orthophosphate inhibition was less severe. There was little or no orthophosphate inhibition in the presence of substrate quantities of 3-phosphoglycerate or ribose 5-phosphate and CO₂.

4. There was also a requirement for phosphate by chloroplasts evolving O₂ in the presence of 3-phosphoglycerate or ribose 5-phosphate plus CO₂. In the presence of endogenous phosphate only, added ribose 5-phosphate suppressed the O₂ evolution which normally followed the addition of 3-phosphoglycerate.

5. The results provide direct support for the proposed phosphate requirement of the photosynthetic carbon cycle and are discussed in this context. They also imply that orthophosphate, ribose 5-phosphate and 3-phosphoglycerate can penetrate the intact chloroplast envelope with considerable rapidity.     

Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO₂ fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO₂ fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110–130 mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO₂ fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.Abbreviations BSA bovine serum albumin - Chl chlorophyll - Pi inorganic orthophosphate - PPi inorganic pyrophosphate - PGA 3-phosphoglycerate - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(aminoethyl ether) - N,N tetraacetic acid     

Carbon dioxide and the reduction of indophenol and ferricyanide by chloroplasts

Pea chloroplasts isolated in salt media show decreased rates of 2:6 dichlorophenolindophenol (DCPIP) and ferricyanide reduction when depleted of CO₂ at pH values below 7.5. The greatest effect of CO₂ was on uncoupled systems. The incorporation of 10⁻², 2 × 10⁻² and 4 × 10⁻² m sodium acetate into the reaction mixtures progressively increased the bicarbonate concentration required for half maximal rates of reduction of DCPIP. The reaction was saturated by bicarbonate concentrations of 1 to 4 × 10⁻² m. With both DCPIP and ferricyanide, the addition of bicarbonate to illuminated chloroplast systems depleted of CO₂ gave very rapid increases in the rates of reduction. Bicarbonate also stimulated oxygen uptake by the illuminated chloroplasts when added hydrogen acceptors had been reduced. There was no effect of bicarbonate on ferricyanide reduction at low light intensities, but with DCPIP reduction, the apparent magnitude of the effect was independent of light intensity. This suggests that DCPIP reacts with the chloroplast electron transport chain at a site nearer to a photochemical stage than does ferricyanide. It also suggests that CO₂ has at least 2 sites of action.     

Regulation of photosynthesis in isolated spinach chloroplasts during orthophosphate limitation

The stromal concentration of orthophosphate in intact spinach chloroplasts (prepared in the absence of orthophosphate or pyrophosphate but supplied with both in the reaction medium) fell from a value of approx. 20 mM in the dark to a steady-state concentration of approx. 8 mM in the light. Chloroplasts illuminated in the absence of orthophosphate or pyrophosphate showed a similar trend. However, in this situation the stromal inorganic phosphate (P_i) concentration rapidly decreased from approx. 10 mM in the dark to a constant steady-state concentration of between 1.5 and 2.5 mM in the light. This P_i concentration was not further diminished (even though CO₂-dependent O₂ evolution had ceased) and was therefore considered to be stromal orthophosphate not freely available to metabolism. In the P_i-deficient chloroplasts the rate of photosynthesis declined rapidly after 1–2 min in the light such that CO₂-dependent O₂ evolution ceased with 5 min of the onset of illumination. The decline in O₂ evolution was accompanied by an increase in the transthylakoid ΔpH (as measured by 9-aminoacridine fluorescence quenching) and in the high-energy state, non-photochemical component of chlorophyll fluorescence quenching (q_E). Measurements of stromal metabolite concentrations showed that the ATP/ADP ratio was decreased in the P_i-deficient chloroplasts relative to chloroplasts illuminated in the presence of P_i. The stromal concentration of glycerate 3-phosphate was comparable in the P_i-deficient chloroplasts and those to which P_i had been supplied. Chloroplasts which were illuminated in P_i-free media showed a large accumulation of ribulose-1,5-bisphosphate relative to those supplied with P_i, suggesting inhibition of ribulose-1,5-bisphosphate carboxylase under these conditions. When P_i was added to chloroplasts illuminated in the absence of P_i, both non-photochemical quenching (q_E), photochemical quenching (q_Q) and ΔpH increased. This suggests that electron transport was not limited by inability to discharge transthylakoid ΔpH. These observation are consistent with the hypothesis that P_i limitation results in decreased ATP production by the thylakoid ATP synthase. The data presented here show that there are multiple sites of flux control exerted by low stromal P_i in the chloroplast. At least three factors contribute to the inhibition of photosynthesis under phosphate limitation: (1) there appears to be a direct effect of P_i on the energy-transducing system; (2) there is direct inhibition of the Calvin cycle decreasing the ability of the pathway to act as a sink for ATP and NADPH; and (3) feedback inhibition of primary processes occurs either via ΔpH or the redox state of electron carriers. However, ΔpH does not appear to be a limiting factor, but rather an inability to regenerate NADP as electron acceptor is suggested. The addition of DCMU to chloroplasts during illumination in the absence of P_i for periods of up to 10 min showed that there was very little loss of variable fluorescence despite a 60% reduction in the capacity for O₂ evolution. This would suggest that photoinhibitory damage to Photosystem II was not the major cause of the inhibition of photosynthesis observed with low P_i.     

The oxidation of C-550 by exogenously added oxidants in the presence of dichlorophenyldimethylurea: The localization of the primary electron acceptor of Photosystem II in the thylakoid membrane

The oxidation of C-550 by exogenously added oxidants in spinachchloroplasts and digitonin-treated chloroplasts was studiedin the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inan attempt to elucidate localization of the primary electronacceptor of Photosystem II in the thylakoid membrane. C-550was directly oxidized by various oxidants such as ferricyanide,N-methylphenazonium methosulfate (PMS) and quinones with redoxpotentials higher than that of C-550. Among the oxidants used,dibromothymoquinone was the most effective oxidant of C-550,followed by PMS. In spite of its high redox potential, ferricyanidewas rather a poor oxidant. The rates of C-550 oxidation by quinoneswere linearly proportional to the oxidant concentration, whereasthe rates tapered off with increasing concentrations of polaroxidants within the ranges of concentration used. These resultsindicate that C-550 is located inside the thylakoid membraneand is covered by a lipophilic shield. Addition of cations,especially divalent cations, significantly affected C-550 oxidationby ferricyanide or PMS but not by nonpolar oxidants. C-550 oxidationby ferricyanide was accelerated at low pH. Thus the accessibilityof C-550 to polar oxidants appears to be affected by electrostaticinteractions between oxidant ions and the negative charges ofthe thylakoid surface. When electrostatic interaction was minimized,ferricyanide oxidized C-550 as rapidly as several quinones did.This suggests that C-550 is located close to the surface ofthe membrane. The evidence indicates that the rates of C-550oxidation depend not only on the accessibility of C-550 to addedoxidants, but also on the reactivity between them. Reduction of cytochrome f by added reductants shows featuressimilar to those of C-550 oxidation by added oxidants, indicatingthat properties of the shield covering cytochrome f are similarto those of the shield covering C-550. (Received March 8, 1977; )     

Manganese content, fluorescence yields and the effect of chloride ion on Hill reaction activity of Tris-washed and reactivated chloroplasts

Reversible Tris-inhibition and reductive reactivation of oxygenevolution activity, observed previously in spinach, were studiedin chloroplasts from Japanese-radish, pokeweed (Phytolacca americana),oats and Easter-lily (Lilium longiflorum). Mn content of Tris-washed and reactivated chloroplasts onlymoderately decreased. Inhibition caused by Tris treatment wasalways greater than the decrease in Mn content, suggesting thatinhibition might not be principally due to Mn decrease. Reactivation enhanced the depressed chlorophyll fluorescencein Tris-inhibited chloroplasts to original levels. Chloride ion promoted oxygen evolution in reactivated, as wellas intact, chloroplasts. Reactivated chloroplasts could perform photophosphorylation,but ammonium ion, which promotes oxygen evolution by uncoupling,did not seem to affect them. (Received November 1, 1971; )     

The Relation between Photophosphorylation and Delayed Light Emission in Chloroplasts

One millisecond delayed light emission has been studied in isolated coupled lettuce (Lactuca sativa var. romaine) chloroplasts. Delayed light emission was increased upon addition of ferricyanide or 2,3,5,6-tetramethyl-p-phenylene diamine. In the presence of ferricyanide, the magnitude of the signal was increased by the addition of ADP (in the absence of orthophosphate), ATP, DI0-9, or phlorizin. The signal was also increased by the addition of NaCl and by the addition of NH₄Cl in the presence of a high NaCl concentration. The signal of delayed light emission was decreased by the addition of gramicidin, valinomycin, and by the addition of NH₄Cl in the presence of a low NaCl concentration.     

Hydrogen peroxide-dependent oxidation of flavonols by intact spinach chloroplasts

Externally added quercetin (100 micromolar) was oxidized by intact spinach chloroplasts at a rate of 30 micromoles per mg chlorophyll per hour in the presence of 100 micromolar H₂O₂. The oxidation rate was increased by about 20% in a hypotonic reaction mixture. The thylakoid fraction also oxidized the flavonol in the presence of H₂O₂, and the rate was about 25% of that by intact chloroplasts. The oxidation of quercetin was inhibited by KCN and NaN₃. Ascorbate, which permeates slowly across chloroplast envelope, only slightly suppressed the initial rate of quercetin oxidation by intact chloroplasts, while the oxidation by ruptured chloroplasts was suppressed by ascorbate by about 60%. Quercetin glycosides, quercitrin and rutin, were also oxidized by chloroplasts in the presence of H₂O₂. These results suggest that flavonols are oxidized by peroxidase-like activity in chloroplasts and that externally added flavonols can permeate into the stroma through the envelope of intact chloroplasts.     

Isolation of Functionally Intact Rhodoplasts from Griffithsia monilis (Ceramiaceae, Rhodophyta)

A procedure is described for isolating photosynthetically active rhodoplasts (“red algal chloroplasts”) from the marine alga Griffithsia monilis. The rhodoplasts exhibited rates of CO₂ fixation and CO₂-dependent O₂ evolution in the order of 200 micromoles per milligram chlorophyll a per hour when illuminated with red or green light and were approximately 80% intact. The response of the rate of photosynthesis to the inorganic phosphate and pyrophosphate concentrations in the medium was qualitatively similar to that previously reported for spinach chloroplasts. Osmotically shocked rhodoplasts evolved O₂ from ferricyanide in red, but not in green, light and were completely uncoupled. Rhodoplast envelope rupture appeared to be accompanied by phycobilisome loss from the thylakoids.     

Bacteriology of Manganese Nodules: V. Effect of Hydrostatic Pressure on Bacterial Oxidation of MnII and Reduction of MnO2

It was experimentally demonstrated that two strains of Arthrobacter 37, one growing at 25 C and the other at 5 C, could catalyze Mn^II oxidation at hydrostatic pressures well in excess of the pressure encountered by the parent culture in its original habitat in the ocean (80 atm). The strain grown at 5 C showed an increase in temperature optimum for manganese oxidation with increase in pressure. It was like-wise experimentally shown that induced Bacillus 29 without added ferricyanide and uninduced Bacillus 29 with added ferricyanide could catalyze MnO₂ reduction at hydrostatic pressures in excess of the pressure encountered by this organism in its original habitat (187 atm). The uninduced Bacillus 29, in the presence of ferricyanide, was active over a wider range of pressures (1 to 1,000 atm) than the induced Bacillus 29 in the absence of ferricyanide (1 to 467 atm). At corresponding pressures, the uninduced culture was also considerably more active than the induced culture. Special techniques were developed for measuring Mn^II-oxidizing and MnO₂-reducing activity under pressure.     

The oxidation of extracellular NADH by sugarcane cells: Coupling to ferricyanide reduction,oxygen uptake and pH change

Suspension-cultured cells of sugarcane (Saccharum sp. hybrids) did not oxidize exogenously supplied NADH in the absence of ferricyanide (potassium hexacyanoferrate [III]), whereas they did at a low rate in the presence of ferricyanide. Concomitantly, ferricyanide was reduced at a slow rate. Neither a pH change nor a change in respiration was caused by the addition of NADH and-or ferricyanide, but ferricyanide was a strong inhibitor of sugar transport. In contrast to cells, protoplasts rapidly oxidized exogenous NADH. This oxidation was accompanied by an increase in oxygen consumption and a net proton disappearance from the medium. Exogenous ferricyanide was reduced only slowly by protoplasts. Simultaneous presence of NADH and ferricyanide produced two effects: 1) a very rapid stoichiometric oxidation of NADH and reduction of ferricyanide until one of the reaction compounds was exhausted, and 2) a nearly instantaneous inhibition of the slower phase of NADH oxidation, which was observed in the presence of NADH but absence of ferricyanide. The extra oxygen consumption and the alkalinization of the medium, as observed with NADH, were also immediately stopped by ferric ions and ferrous ions. The presence of NADH and ferricyanide caused a fast stoichiometric acidification of the medium. These results were taken as evidence that the oxidation of NADH in the absence of ferricyanide is not related to the NADH-ferricyanide-coupled redox reaction. Furthermore, addition of NADH caused some uncoupling of the protoplasts, an effect which would explain the strong acidification of the cell cytoplasm and the inhibition of various transport systems. The NADH-oxidizing systems oxidized both the -configurated pyridine nucleotide and the -configurated form. Since NADH-linked dehydrogenases usually do not work with -NADH (with the exception of the endoplasmic-reticulum-bound electron-transport system), the observed activities could have been derived from contaminating membranes and dying protoplasts in the suspension. All reported reactions partly or predominantly occurred in the supernatant of the protoplast suspension and increased considerably during incubation of the protoplasts. The rates and quantities of oxygen consumption, pH change, and ferricyanide reduction fitted with NADH oxidation in a stoichiometric ratio, which implied that all these reactions occurred in the extracellular space, without involving transmembrane steps. No evidence for a physiological role in energization of the plasmalemma was found.Abbreviation NADH -nicotinamide adenine dinucleotide reduced form     

Glycolate synthesis by intact chloroplasts: Studies with inhibitors of photophosphorylation

Intact spinach chloroplasts, capable of evolving O₂ in response to CO₂ at rates greater than 70 μmol/h · mg of chlorophyll, synthesize glycolate from added dihydroxyacetone phosphate, ribose 5-phosphate, or xylulose 5-phosphate, when illuminated in the presence of O₂. The synthesis of glycolate from these compounds is dependent upon photophosphorylation and is inhibited by each of the three classes of photophosphorylation inhibitors [Izawa, S., and Good, N. E. (1972) in Methods in Enzymology, Vol. 24, Part B, pp. 355–377)]: an uncoupler, carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP), an energy transfer inhibitor, Dio-9, and a phosphate analog, arsenate. Neither arsenate nor Dio-9 causes the collapse of the light-generated proton gradient between thylakoid and stroma compartments of the chloroplasts, so that the inhibition of glycolate synthesis by these compounds cannot be ascribed to an inactivation of Calvin cycle enzymes thought to be associated with the collapse of such a proton gradient. The dependency of glycolate synthesis upon photophosphorylation indicates that an ATP-consuming reaction(s) is involved in the conversion of the substrates (triose and pentose monophosphates) to glycolate. The formation of dihydroxyethylthiamine pyrophosphate, the “active glycolaldehyde” intermediate of the transketolase reaction, from triose and pentose monophosphates has no known requirements for ATP. On the other hand, the conversion of both triose and pentose monophosphates to ribulose 1,5-bisphosphate, the substrate for the ribulose 1,5-bisphosphate oxygenase reaction, requires ATP. It is concluded that glycolate synthesis by intact isolated chloroplasts is primarily the result of ribulose 1,5-bisphosphate oxygenase activity. No substantial role in glycolate synthesis can be attributed to the oxidation of dihydroxyethylthiamine pyrophosphate, the intermediate of the transketolase reaction.     

Unidirectional transport of orthophosphate across the envelope of isolated cauliflower-bud amyloplasts

Isolated amyloplasts from cauliflower (Brassica oleracea L. var botrytis) buds are able to export orthophosphate unidirectionally into the incubation medium. This orthophosphate transport appears to be protein-mediated, as indicated by the following observations: (i) low temperature and the presence of inhibitors of protein-mediated transport reduced the rate of orthophosphate export, and (ii) the rate of orthophosphate export became saturated with rising internal substrate concentrations. Micromolar concentrations of 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid inhibited the rate of unidirectional orthophosphate export, thus indicating the involvement of the amyloplastic glucose-6-phosphate (Glc6P)translocator in the unidirectional export of orthophosphate. The effect of rising concentrations of orthophosphate upon the activity of ADP glucose pyrophosphorylase in desalted extracts was determined. Orthophosphate given in concentrations similar to those measured in the amyloplastic stroma under conditions of steady-state rates of Glc6P-dependent starch synthesis inhibited the activity of ADP-glucose pyrophosphorylase significantly. However, even under strong limiting substrate conditions the residual activity was sufficient to catalyze the flux of carbon into starch. The maximal rates of orthophosphate transport (in the counter-exchange mode) by isolated spinach (Spinacia oleracea L.) chloroplasts and by isolated cauliflower-bud amyloplasts were also determined. These rates were compared with the maximal rates of undirectional orthophosphate export by these plastids. From these measurements we can conclude that, compared with spinach chloroplasts, isolated amyloplasts of cauliflower exhibit a fivefold greater ratio of unidirectional orthophosphate transport to maximal rate of orthophosphate transport in the counter-exchange mode compared to spinach chloroplasts. The determined rate of maximal unidirectional orthophosphate export is sufficient to catalyze the release of additional inorganic phosphate liberated in the amyloplastic stroma during the process of Glc6P-dependent starch synthesis.     

Production of Metallogenium-like particles by heterotrophic manganese-oxidizing bacteria collected from a lake

As a model of chemically stratified structure of environment typical to the chemocline of lakes, a semisolid gradient medium was prepared to cultivate heterotrophic manganese-oxidizing bacteria originally collected from a lake. The bacteria growing under the conditions described produced extracellularly Metallogenium-like particles similar to those which are often detected in the chemocline of lakes. This suggested that the naturally occurring Metallogenium-like particles originated in activities of such heterotrophic manganese-oxidizing bacteria. The manganese oxidation activity usually appeared only at the stationary phase of bacterial growth. The oxidation of Mn²⁺ and the formation of Metallogenium-like particles by the bacteria were observed at neutral or slightly acidic pH. not under alkaline conditions, which is a conspicuous difference from the inorganic oxidation of Mn²⁺ by O₂. Bacterial manganese oxidation was stimulated by bicarbonate (50 or 500 M). An experiment of addition of H₂O₂ to the incubation tubes isolated from atmosphere failed to confirm the availability of externally added H₂O₂ as the electron acceptor, suggesting that the bacterial manganese oxidation required the presence of O₂.     

Regulation of horse-liver glutathione reductase

• 1.1. The enzyme was rapidly inactivated by NAD(P)H, GSH, dithionite or borohydride, while activity increased in the presence of NAD(P)+ or GSSG. NADH was more efficient for inactivation than NADPH. Redox inactivation required neutral or alkaline pH, was maximal at pH 8.5, and depended on the presence of metal cations.
• 2.2. GSSG and dithiothreitol fully protected the enzyme from inactivation at concentrations stoichiometric with NAD(P)H. Ten-fold higher ferricyanide or GSH concentrations were required to obtain partial protection. NAD+ or NADP+ were quite ineffective.
• 3.3. GSSG fully reactivated the inactive enzyme at 38°C and neutral to acidic pH (5.5–7.5). Reactivation by dithiothreitol was accomplished in short periods of time at pH 8.5 although the activity was progressively lost afterwards. Ferricyanide and GSH also reactivated the enzyme to different extents.

     

Light-dependent reduction of pyruvate by pea chloroplasts in the presence of glutamate dehydrogenase and C5-dicarboxylic acids

Illuminated pea chloroplasts supported (glutamine plus α-oxoglutarate (α-OG)) and (NH₃ plus α-OG)-dependent O₂ evolution. The properties of these reactions were consistent with light-coupled glutamate synthase and glutamine synthetase activities. In the presence of a glutamate-oxidizing system (component C) comprised of NAD-specific glutamate dehydrogenase (NAD-GDH), lactate dehydrogenase (LDH), 4 mM pyruvate and 0.2 mM NAD, illuminated chloroplasts supported O₂ evolution in the presence of glutamine. The reaction did not proceed in the absence of any one of the constituents of component C and the properties of O₂ evolution were consistent with light-coupled glutamate synthase activity. In the presence of component C, chloroplasts also catalysed O₂ evolution in the presence of catalytic concentrations of glutamate. Studies of O₂ evolution and metabolism of [¹⁴C]-glutamate in the presence of the inhibitors methionine sulphoximine (MSO) and azaserine suggest that O₂ evolution was dependent on the synthesis of glutamine from the products of glutamate oxidation. This was supported by polarographic studies using α-OG and NH₃ instead of glutamate.The results are consistent with a C₅-dicarboxylic acid shuttlemechanism for the export of reducing equivalents from illuminated chloroplasts (glutamate) and recycling of the oxidation products (α-OG and NH₃).     

Photosynthetic oxygen evolution and chlorophyll fluorescence in intact isolated chloroplasts on a solid support: the influence of orthophosphate

We devised recently a method to trap intact isolated chloroplasts on a solid support consisting of membrane filters made of cellulose nitrate (Cerovi et al., 1987, Plant Physiol. 84, 1249–1251). The addition of alkaline phosphatase to the reaction medium enabled continuous photosynthesis by spinach (Spinacia oleracea L.) chloroplasts to be sustained by hydrolysis of newly produced and exported triose phosphates and recycling of orthophosphate. In this system, simultaneous measurements of chlorophyll fluorescence and oxygen evolution were performed and their dependence on orthophosphate concentration was investigated. Optimal photosynthesis was obtained at a much higher initial orthophosphate concentration (2–4 mM) compared to intact chloroplasts in suspension. Secondary kinetics of chlorophyll fluorescence yield were observed and were shown to depend on the initial orthophosphate concentration.Abbreviations Chl chlorophyll - CSS intact isolated chloroplasts on solid support - ICS intact isolated chloroplasts in suspension - P_i orthophosphate - v rate of O₂ evolution - PPFD photosynthetic photon flux density The authors wish to thank Dr. Marijana Plesniar, from the University of Novi Sad, for stimulating discussions. This work was supported by the Fond for Science of the Republic of Serbia. Z.G.C.'s visit to the Robert Hill Laboratory was supported by the British Council and the University of Sheffield.     

Oxidation of manganous pyrophosphate by superoxide radicals and illuminated spinach chloroplasts.

The oxidation of Mn²⁺-pyrophosphate to Mn³⁺ by superoxide (O₂^?) was quantitative as evidenced from the formation of Mn³⁺-pyrophosphate and hydrogen peroxide and from the inhibition by superoxide dismutase. Using the competitive relation between Mn²⁺-pyrophosphate and superoxide dismutase for the O₂^?, the rate constant of Mn²⁺ oxidation was estimated to be about 6 × 10⁶m^?1 s^?1. The oxidation of Mn²⁺-pyrophosphate by illuminated chloroplasts was also indicated to be stoichiometrically induced by O₂^?. In the presence of saturating amounts of the Mn²⁺, a double enhancement of hydrogen peroxide production and triple uptake of oxygen were found, as expected from the oxidation of Mn²⁺-pyrophosphate by O₂^?. Anaerobiosis or superoxide dismutase annuled these increments. We propose that the O₂^? generated as the sole initial step of the Mehler reaction oxidized Mn²⁺-pyrophosphate, and we discuss the role of free manganese in chloroplasts.