Pyrophosphate inhibition of carbon dioxide fixation in isolated pea chloroplasts by uptake in exchange for endogenous adenine nucleotides

Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum) chloroplasts was inhibited by inorganic pyrophosphate (PPi). Oxygen evolution was also inhibited by high concentrations of orthophosphate (Pi) and the inhibition was relieved by 3-phosphoglycerate. In contrast, the inhibition by PPi was not relieved by 3-phosphoglycerate, indicating that hydrolysis of PPi and accumulation of inhibitory concentrations of Pi were not occurring. In agreement with this suggestion, the percentage of (14)C-labeled products diffusing out of the chloroplasts was increased by Pi but not by PPi. The inhibition of O(2) evolution by PPi was reversed by ATP. The concentration of PPi required for 50% inhibition was 1.2 to 1.4 mm and the subsequent stimulation by ATP was half-maximal at 16 to 25 mum. Carbon dioxide-dependent O(2) evolution by spinach chloroplasts, or chloroplasts isolated from older pea plants, was not significantly inhibited by PPi.Chloroplasts were preloaded with (14)C-ATP and release of the labeled nucleotides was measured to assess the activity of adenine nucleotide transport across the inner chloroplast envelope membrane. A rapid exchange was promoted by the addition of exogenous ATP. Addition of PPi also resulted in a release of endogenous nucleotides. We suggest that PPi inhibits CO(2) fixation by entering the chloroplast in exchange for endogenous adenine nucleotides via the transporter on the inner envelope membrane. The subsequent depletion of the internal adenine nucleotide pool would result in decreased CO(2) fixation due to insufficient ATP. Addition of ATP to PPi-inhibited chloroplasts apparently results in uptake of catalytic amounts of ATP and restoration of the internal adenine nucleotide pool thus relieving the inhibition of CO(2) fixation.     

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.     

Silicomolybdate reduction by isolated pea chloroplasts

Conditions for the optimization of silicomolybdate reduction by isolated pea chloroplasts are described. Maximum rates of reduction are related to time of addition to the chloroplasts and the presence of an oxidizing cofactor, such as ferricyanide. Silicomolybdate or silicomolybdate plus ferricyanide reduction is only partially inhibited by a concentration of CMU which totally abolishes ferricyanide reduction. Evidence for a differing response of the two reduction sites to silicomolbydate is described.     

Photosynthetic control by isolated pea chloroplasts

Isolated pea chloroplasts undergo both cyclic and non-cyclic electron flow. Both processes are coupled to photophosphorylation. During non-cyclic flow the rate of oxygen production showed ADP-governed ;photosynthetic control' analogous to respiratory control of isolated mitochondria. Measurements of ADP/O and photosynthetic control ratios yielded values of 1-1.3 and 2-5.7 respectively. ;Photosynthetic control' was shown to be dependent on the intactness of the chloroplasts.     

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.     

Carbon dioxide fixation by immobilized chloroplasts

Chloroplasts of chinese mustard (Brassica campestris L.) were immobilized in polyacrylamide gel. A 8% polymer concentration was suitable for the immobilization. The activity of the carbon dioxide fixation of immobilized chloroplasts was 65% of that of free chloroplasts. The optimum conditions for the carbon dioxide fixation of immobilized chloroplasts were similar to that of native chloroplasts. However, immobilized chloroplasts were more stable under alkaline conditions and high temperatures than native chloroplasts. Light penetration of the gel was not a limiting parameter of the carbon dioxide fixation. The lifetime of immobilized chloroplasts was three times longer than that of free chloroplasts. 3-Phosphoglyceraldehyde and other compounds were produced continuously by immobilized chloroplasts.     

Photosynthesis by isolated chloroplasts. Inhibition by dl-glyceraldehyde of carbon dioxide assimilation

Photosynthetic carbon assimilation and associated CO(2)-dependent O(2) evolution by chloroplasts isolated from pea shoots and spinach leaves is almost completely inhibited by 10mm-dl-glyceraldehyde. The inhibitor is without appreciable effect on photosynthetic electron transport, photophosphorylation, the carboxylation of ribulose 1,5-diphosphate or the reduction of 3-phosphoglycerate, but apparently blocks the conversion of triose phosphate into ribulose 1,5-diphosphate.     

Stimulation of human neutrophil adhesive properties by adenine nucleotides

Inasmuch as adenine nucleotides may be secreted by platelets during inflammation, we sought to determine whether ATP and related compounds could serve as stimuli of neutrophil (polymorphonuclear cells, PMN) activation as manifested by an increase in their adhesive properties. Exposure of isolated human PMN to ATP or its nonhydrolyzable analog, adenosine 5'-O-(3-thiotriphosphate) did indeed stimulate an increase in cellular adhesive function as assessed by an increase in the surface expression of the leukocyte adhesion-promoting glycoprotein, Mo1 (CD11b/CD18), the initiation of PMN aggregation, and (in the case of ATP) the attachment of increased numbers of albumin-coated polystyrene latex beads. However, this increase in PMN adhesive function was not accompanied by the generation of products of the respiratory burst. These in vitro data suggest the possible influence of secreted adenine nucleotides in promoting neutrophil adhesion-dependent interactions at inflammatory sites in vivo.