Stimulation of carbon dioxide fixation in isolated pea chloroplasts by catalytic amounts of adenine nucleotides

Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum var. Massey Gem) chloroplasts was increased two to 12 times by the addition of ATP. O(2) evolution was also stimulated by ADP and to a lesser extent by AMP. The ATP effects were not due to broken chloroplasts present in the preparations nor was ATP acting as a phosphate source. We concluded that the adenine nucleotides were acting catalytically. The concentration of ATP required for half-maximum rate of O(2) evolution was 16 to 25 mum. The degree to which ATP stimulated O(2) evolution depended on the age of pea plants from which the chloroplasts were isolated. Spinach (Spinacia oleracea var. True Hybrid 102) chloroplasts did not show a consistent stimulation of O(2) evolution by adenine nucleotides.The adenine nucleotide content of pea chloroplasts was not lower than that of spinach chloroplasts, but pea chloroplasts which showed a large stimulation of O(2) evolution by ATP contained an ATP-hydrolyzing reaction with rates of 10 to 50 mumol ATP hydrolyzed mg chlorophyll(-1) hour(-1). The rate of the ATP-consuming reaction was much lower in spinach chloroplasts and in chloroplasts from older pea plants which did not show large stimulation of O(2) evolution by ATP. We propose that the ATP-consuming reaction, with a high affinity for ATP, decreased the effective size of the ATP pool available for CO(2) fixation. Added adenine nucleotides could be transported into the chloroplasts increasing the concentration of internal nucleotides. Calculations showed that the adenine nucleotide transporter on the outer chloroplast membranes could operate at a sufficient rate to produce such an effect.     

Light-enhanced carbon dioxide fixation in isolated chloroplasts

Preillumination of leaves of spinach, soybean and maize in theabsence of CO₂ greatly enhanced the capacity for fixing CO₂in an immediately following dark period. Lightenhanced darkCO₂-fixation was further observed in isolated chloroplasts ofspinach and soybean. When isolated chloroplasts were illuminated,CO₂-fixing capacity in the subsequent dark period increasedrapidly at first and later more slowly attaining a stationaryvalue in about 20 min. When the light was turned off at thisstage, the capacity decreased very rapidly becoming zero inabout 10 min. The magnitude of the enhanced dark fixation andits decay in the dark were not influenced by the presence orabsence of atmospheric oxygen. In both leaves and isolated chloroplasts,no significant change in oxygen (21%) occurred in distributionpatterns of radioactivity in products fixed by photosynthetic,or light-enhanced, dark, ¹⁴CO₂-fixation. In preilluminated leaves¹⁴C was incorporated into sucrose in the subsequent dark period,indicating that the photosynthetic carbon reduction cycle isoperating in light-enhanced dark fixation in higher plants. ¹Present address: Noda Institute for Scientific Research, Noda,Chiba Prefecture (Received August 10, 1970; )     

Energetic factors affecting carbon dioxide fixation in isolated chloroplasts

Light- and HCO₃⁻-saturated (10 millimolar) rates of O₂ evolution (120 to 220 micromoles O₂ per milligram chlorophyll per hour), obtained with intact spinach chloroplasts, are decreased up to 3-fold by changes in assay conditions such as omission of catalase from the medium, the use of high (≥1 millimolar) inorganic phosphate, inclusion of NO₂⁻ as an electron acceptor, or bright illumination at low partial pressures of O₂. These inhibitions may be reversed by addition of uncoupling levels of NH₄Cl or of antimycin concentrations that partially block cyclic electron transfer between cytochrome b₆ and cytochrome f. Measurements of the pH gradient across the thylakoid membrane with the fluorescent probe, 9-aminoacridine, indicate that changes in ΔpH are sufficient to account for both the inhibited and restored rates of electron transport. It follows that the rate of HCO₃⁻-saturated photosynthesis may be restricted by a proton gradient back pressure under these conditions.     

Carbon dioxide fixation by chloroplasts isolated in glycinebetaine

Spinach chloroplasts capable of high rates of CO₂ fixation have been isolated in glycinebetaine as an alternative osmoticum to sorbitol and found to be very stable. Proline was a less satisfactory alternatine. The possible significance of the use of glycinebetaine is discussed as this solute may be the physiological cytoplasmic osmoticum in members of the Chenopodiaceae.     

Effects of glycidate on carbon dioxide fixation with isolated spinach chloroplasts

Glycidate (2,3-epoxypropionate) stimulated CO₂ fixation in isolated spinach chloroplasts up to 100%. In the presence of glycidate the initial lag phase was abolished and the chloroplasts exported mainly 3-phosphoglycerate instead of dihydroxyacetone phosphate.     

Carbon dioxide fixation in isolated kalanchoe chloroplasts

Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 μmoles of CO₂ per milligram of chlorophyll per hour. The dark rate of fixation was about 1% of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1,6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO₂ fixation were primarily those of the photosynthetic carbon reduction cycle.     

Some effects of sugars and sugar phosphates on carbon dioxide fixation by isolated chloroplasts

1. Carbon dioxide fixation by isolated pea chloroplasts was stimulated by the addition of intermediates of the Calvin photosynthesis cycle and by some related compounds. 2. Ribose 5-phosphate and fructose 1,6-diphosphate consistently produced the largest effects; free sugars such as erythrose and sedoheptulose and acids such as glycollate and glyoxylate were largely ineffective or even inhibitory. 3. Small effects were produced by fructose and ribose but not by their isomers, glucose and xylose. 4. Maximal rates in the presence of ribose 5-phosphate varied between 10 and 50μmoles of carbon dioxide fixed/mg. of chlorophyll/hr.     

Calvin-cycle intermediates in relation to induction phenomena in photosynthetic carbon dioxide fixation by isolated chloroplasts

1. Induction periods in carbon dioxide fixation by isolated pea chloroplasts were shortened by small quantities of Calvin-cycle intermediates. The additional fixation was larger than that which would have followed direct stoicheiometric conversion into ribulose 1,5-diphosphate. 2. When chloroplasts were illuminated in the absence of added substrates (other than carbon dioxide) soluble products were formed in the medium that stimulated fixation by fresh chloroplasts. 3. The induction periods were lengthened by washing the chloroplasts. Addition of catalytic quantities of Calvin-cycle intermediates then decreased the induction periods to their previous values. 4. The induction period was extended by a decrease in temperature but was largely unaffected by a decrease in light-intensity that was sufficient to decrease the maximum rate. 5. It is concluded that the lag periods are a consequence of the loss of Calvin-cycle intermediates, such as sugar phosphates, through the intact chloroplast envelope and that these losses can be made good by new synthesis from carbon dioxide in the reactions of the Calvin cycle.     

Influence of adenine nucleotides on the inhibition of photophosphorylation in spinach chloroplasts by N-ethylmaleimide.

The incubation of spinach chloroplasts with 1 mM N-ethylmaleimide in light for 60 to 90 s results in a partial, irreversible inhibition of photophosphorylation. The inhibition was not overcome at infinite light intensity or at infinite concentrations of the phosphorylation substrates. Although the inhibition diminished with decreasing concentrations of adenosine diphosphate in the assay of phosphorylation, the inhibition of guanosine diphosphate phosphorylation was independent of the concentration of this nucleotide. Although adenosine di- or triphosphate (10 to 30 muM) alone partially prevented the development of the N-ethylmaleimide inhibition of phosphorylation, these nucleotides were more effective when either 1 mM inorganic phosphate or arsenate was also present. The light-dependent incorporation of N-ethylmaleimide into chloroplast-bound coupling factor 1 was affected by adenosine triphosphate and inorganic phosphate in a manner similar to the onset of N-ethylmaleimide inhibition. Since guanosine diphosphate did not protect phosphorylation from N-ethylmaleimide inhibition but is phosphorylated at rapid rates, it is apparent that coupling factor 1 in chloroplasts has multiple nucleotide recognition sites.     

Sulfur dioxide inhibition of photosynthesis in isolated spinach chloroplasts

Photosynthetic oxygen evolution by isolated spinach (Spinacia oleracea L.) chloroplasts approached complete inhibition in the presence of a 5 mm concentration of sulfur dioxide. A similar inhibition was observed in the presence of equimolar concentrations of bisulfite ions, suggesting a parallel mode of action. In contrast, an equimolar concentration of sulfite ions was markedly less inhibitory and sulfate ions caused negligible inhibition of apparent photosynthesis. The mode of action of sulfur dioxide and related sulfur anions in inhibiting photosynthesis was found to be essentially independent of direct hydrogen-ion effects. Supplements of inorganic pyrophosphate lessened the inhibition of oxygen evolution caused by sulfur dioxide and the sulfur anions.Sulfur dioxide and the sulfur anions were almost equally effective in inhibiting cyclic and noncyclic photophosphorylation in chloroplast suspensions. However, the extent of the inhibition of these photosynthetic reactions does not appear sufficient to account for the inhibition of photosynthetic oxygen evolution by sulfur dioxide.     

Inhibition of photosynthetic carbon dioxide fixation in isolated spinach chloroplasts exposed to reduced osmotic potentials

Reduced osmotic potentials inhibited the rate of CO₂ fixation by isolated intact spinach (Spinacia oleracea) chloroplasts. This inhibition was observed immediately after transfer of chloroplasts from a solution containing 0.33 m sorbitol to higher sorbitol concentrations, and the depressed rate remained constant. The inhibited CO₂ fixation could not be attributed to a decreased rate of photosynthetic electron transport, since NADP reduction was unaffected by subjecting the chloroplasts to low potentials. It could also not result from restricted permeability to CO₂, as CO₂ concentrations had no effect on the relative inhibition induced by the lowered potential.     

Characteristics of light-dependent inorganic carbon uptake by isolated spinach chloroplasts

The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach (Spinacia oleracea L.) chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5- to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5°C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [¹⁴C]dimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and ¹⁴CO₂ bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO₂ in the chloroplast.     

Assay of nucleotides and other phosphate-containing compounds in isolated chloroplasts by ion exchange chromatography

This report describes a method for a sensitive assay of phosphate-containing metabolites in chloroplasts. The first step was to preincubate the chloroplasts with inorganic [³²P]phosphate in order to label the stromal phosphate pool. On illumination, the intermediates of the reductive CO₂ fixation cycle, and also ADP and ATP, were radioactively labeled. After a metabolic stop by filtering centrifugation into perchloric acid, the resulting extract was chromatographed on a small ion exchange column with a formic acid-formate gradient. The separated substances which appeared in the eluate were detected by continuous monitoring of ³²P radioactivity. The identity of the peaks in the chromatogram and the reproducibility of the assay procedure were investigated.     

Stoichiometry of proton uptake in isolated pea chloroplasts under different light intensities

The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H⁺/e^– ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W · m^–2) (strong light) the H⁺/e^– ratio was 3. At low intensity (0.9 W · m^–2) (weak light) the H⁺/e^– ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H⁺/e^– ratio due to underestimation of the initial rate of H⁺ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative.